SUBSTITUTED PYRAZOLO[3,4-d]PYRIMIDINES AS WEE1 INHIBITORS

ABSTRACT

This invention provides for substituted pyrazolo[3,4-d]pyrimidine compounds of the Formula (I): 
     
       
         
         
             
             
         
       
     
     as Wee1 inhibitors. The substituted pyrazolo[3,4-d]pyrimidine compounds may find use as therapeutic agents for the treatment of diseases. The substituted pyrazolo[3,4-d]pyrimidine compounds may also find particular use in oncology.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.16/843,713, filed Apr. 8, 2020, which claims priority to U.S.Provisional Application No. 62/831,665, filed on Apr. 9, 2019, thecontents of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

This disclosure relates generally to therapeutics engaged in inhibitionof the DNA damage checkpoint kinase, Wee1, which potentiates genotoxicchemotherapies by abrogating cell-cycle arrest and proper DNA repair.The invention also provides pharmaceutically acceptable compositionscomprising compounds of the present invention and methods of using saidcompositions in the treatment of diseases associated with this pathway.

BACKGROUND OF THE INVENTION

Wee1 is a tyrosine kinase that phosphorylates and inactivates Cdc2 andis involved in G checkpoint signaling. More particularly, Wee1 isinvolved in G₂-M checkpoint signaling. Because p53 is a key regulator inthe G checkpoint, p53-deficient tumors rely only on the G checkpointafter DNA damage. More particularly, because p53 is a key regulator inthe G₁-S checkpoint, p53-deficient tumors rely only on the G₂-Mcheckpoint after DNA damage. Hence, such tumors are selectivelysensitized to DNA-damaging agents by Wee1 inhibition.

Wee1 belongs to a family of protein kinases involved in the terminalphosphorylation and inactivation of cyclin-dependent kinase 1-boundcyclin B, resulting in G cell cycle arrest in response to DNA damage.Wee1 was first identified in fission yeast, where Wee1 deficiencyresulted in premature mitotic entry and replication of smaller-sizedyeast. It is the major kinase responsible for the inhibitoryphosphorylation of the tyrosine.

Before cells undergo mitosis, they progress through a tightly controlledcascade of G₁-S, intra-S, and G₂-M checkpoints. Wee1 kinase has emergedas a key G₂-M checkpoint regulator. This tyrosine kinase negativelyregulates entry into mitosis by catalyzing an inhibitory phosphorylationof Cdc2 (the human homolog of cyclin-dependent kinase 1 (CDK1) ontyrosine-15 (Y15). This results in inactivation of the Cdc2/cyclin Bcomplex, which arrests cells in G₂-M, allowing for DNA repair. Suchinhibition also occurs through Chk1-mediated inhibition of Cdc25phosphatases, which remove the inhibitory phosphorylation on Cdc2. Thus,entry into mitosis rests on a balance between the opposing activities ofWee1 and Chk1/Cdc25. Wee1 inhibition is thus expected to abrogate G₂-Marrest and propel cells into premature mitosis, a hypothesis confirmedby studies documenting that Wee1 inhibition by either small moleculeinhibitors or small interference RNA leads to premature entry intomitosis and consequent cell death through mitotic catastrophe orapoptosis. (S. Muller, J. Clinical. Oncology, 2015).

Recently, a few classes of Wee1 inhibitors have been disclosed. Amongthem is a selective inhibitor, AZD-1775 (1,2-allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-((4-(4methylpiperazin-1-yl)phenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one).AZD-1775 exhibited antitumor activity in various preclinical studies asa monotherapy or in potentiating chemo- and radiotherapy, and iscurrently in phase I/II clinical trials.

Wee1 is highly expressed in several cancer types, includinghepatocellular carcinoma, breast cancers, cervical cancers, lungcancers, squamous cell carcinoma, diffuse intrinsic pontine glioma(DIPG), glioblastoma, medulloblastoma, leukemia, melanoma, and ovariancancers. (P. Reigan et al., Trends in Pharmacol. Sci., 2016).

There are few Wee1 inhibitors in clinical development. There is scope toimprove Wee1 inhibitor selectivity and the properties of the inhibitorsto permit targeting of specific cancer types.

BRIEF SUMMARY OF THE INVENTION

In one aspect, provided is a compound of Formula (I):

or a salt thereof, wherein Y, R¹, R², R³, R⁴, m and n are as detailedherein.

In some embodiments, the compound of Formula (I) or a salt thereof, isof the Formula (II) or (III), or a salt thereof as detailed herein.

In another aspect, provided is a method of treating cancer in anindividual in need thereof comprising administering to the individual atherapeutically effective amount of a compound as detailed herein, suchas a compound of Formula (I), (II) or (III) or a pharmaceuticallyacceptable salt thereof. Also provided is a method of inhibiting Wee1 ina cell, comprising administering a compound detailed herein, or a saltthereof, to the cell.

In another aspect, provided are pharmaceutical compositions comprising acompound detailed herein and a pharmaceutically acceptable carrier orexcipient. Kits comprising a compound detailed herein or a salt thereofare also provided. A compound as detailed herein, or a salt thereof, isalso provided for the manufacture of a medicament for the treatment ofcancer.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Alkyl” refers to and includes saturated linear and branched univalenthydrocarbon structures and combination thereof, having the number ofcarbon atoms designated (i.e., C₁-C₁₀ means one to ten carbons).Particular alkyl groups are those having 1 to 20 carbon atoms (a “C₁-C₂₀alkyl”). More particular alkyl groups are those having 1 to 8 carbonatoms (a “C₁-C₈ alkyl”), 3 to 8 carbon atoms (a “C₃-C₈ alkyl”), 1 to 6carbon atoms (a “C₁-C₆ alkyl”), 1 to 5 carbon atoms (a “C₁-C₅ alkyl”),or 1 to 4 carbon atoms (a “C₁-C₄ alkyl”). Examples of alkyl include, butare not limited to, groups such as methyl, ethyl, n-propyl, isopropyl,n-butyl, t-butyl, isobutyl, sec-butyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

“Alkenyl” as used herein refers to an unsaturated linear or branchedunivalent hydrocarbon chain or combination thereof, having at least onesite of olefinic unsaturation (i.e., having at least one moiety of theformula C═C) and having the number of carbon atoms designated (i.e.,C₂-C₁₀ means two to ten carbon atoms). The alkenyl group may be in “cis”or “trans” configurations, or alternatively in “E” or “Z”configurations. Particular alkenyl groups are those having 2 to 20carbon atoms (a “C₂-C₂₀ alkenyl”), having 2 to 8 carbon atoms (a “C₂-C₈alkenyl”), having 2 to 6 carbon atoms (a “C₂-C₆ alkenyl”), or having 2to 4 carbon atoms (a “C₂-C₄ alkenyl”). Examples of alkenyl include, butare not limited to, groups such as ethenyl (or vinyl), prop-1-enyl,prop-2-enyl (or allyl), 2-methylprop-1-enyl, but-1-enyl, but-2-enyl,but-3-enyl, buta-1,3-dienyl, 2-methylbuta-1,3-dienyl, homologs andisomers thereof, and the like.

“Alkylene” as used herein refers to the same residues as alkyl, buthaving bivalency. Particular alkylene groups are those having 1 to 6carbon atoms (a “C₁-C₆ alkylene”), 1 to 5 carbon atoms (a “C₁-C₅alkylene”), 1 to 4 carbon atoms (a “C₁-C₄ alkylene”) or 1 to 3 carbonatoms (a “C₁-C₃ alkylene”). Examples of alkylene include, but are notlimited to, groups such as methylene (—CH₂—), ethylene (—CH₂CH₂—),propylene (—CH₂CH₂CH₂—), butylene (—CH₂CH₂CH₂CH₂—), and the like.

“Alkynyl” as used herein refers to an unsaturated linear or branchedunivalent hydrocarbon chain or combination thereof, having at least onesite of acetylenic unsaturation (i.e., having at least one moiety of theformula C≡C) and having the number of carbon atoms designated (i.e.,C₂-C₁₀ means two to ten carbon atoms). Particular alkynyl groups arethose having 2 to 20 carbon atoms (a “C₂-C₂₀ alkynyl”), having 2 to 8carbon atoms (a “C₂-C₈ alkynyl”), having 2 to 6 carbon atoms (a “C₂-C₆alkynyl”), or having 2 to 4 carbon atoms (a “C₂-C₄ alkynyl”). Examplesof alkynyl include, but are not limited to, groups such as ethynyl (oracetylenyl), prop-1-ynyl, prop-2-ynyl (or propargyl), but-1-ynyl,but-2-ynyl, but-3-ynyl, homologs and isomers thereof, and the like.

“Aryl” refers to and includes polyunsaturated aromatic hydrocarbongroups. Aryl may contain additional fused rings (e.g., from 1 to 3rings), including additionally fused aryl, heteroaryl, cycloalkyl,and/or heterocyclyl rings. In one variation, the aryl group containsfrom 6 to 14 annular carbon atoms. Examples of aryl groups include, butare not limited to, phenyl, naphthyl, biphenyl, and the like.

“Carbonyl” refers to the group C═O.

“Cycloalkyl” refers to and includes cyclic univalent hydrocarbonstructures, which may be fully saturated, mono- or polyunsaturated, butwhich are non-aromatic, having the number of carbon atoms designated(e.g., C₁-C₁₀ means one to ten carbons). Cycloalkyl can consist of onering, such as cyclohexyl, or multiple rings, such as adamantyl, butexcludes aryl groups. A cycloalkyl comprising more than one ring may befused, spiro or bridged, or combinations thereof. A preferred cycloalkylis a cyclic hydrocarbon having from 3 to 13 annular carbon atoms. A morepreferred cycloalkyl is a cyclic hydrocarbon having from 3 to 8 annularcarbon atoms (a “C₃-C₈ cycloalkyl”). Examples of cycloalkyl include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, norbomyl, and the like.

“Halo” or “halogen” refers to elements of the Group 17 series havingatomic number 9 to 85. Preferred halo groups include fluoro, chloro,bromo and iodo. Where a residue is substituted with more than onehalogen, it may be referred to by using a prefix corresponding to thenumber of halogen moieties attached, e.g., dihaloaryl, dihaloalkyl,trihaloaryl etc. refer to aryl and alkyl substituted with two (“di”) orthree (“tri”) halo groups, which may be but are not necessarily the samehalo; thus 4-chloro-3-fluorophenyl is within the scope of dihaloaryl. Analkyl group in which each hydrogen is replaced with a halo group isreferred to as a “perhaloalkyl.” A preferred perhaloalkyl group istrifluoroalkyl (—CF₃—). Similarly, “perhaloalkoxy” refers to an alkoxygroup in which a halogen takes the place of each H in the hydrocarbonmaking up the alkyl moiety of the alkoxy group. An example of aperhaloalkoxy group is trifluoromethoxy (—OCF₃).

“Heteroaryl” refers to and includes unsaturated aromatic cyclic groupshaving from 1 to 10 annular carbon atoms and at least one annularheteroatom, including but not limited to heteroatoms such as nitrogen,oxygen and sulfur, wherein the nitrogen and sulfur atoms are optionallyoxidized, and the nitrogen atom(s) are optionally quaternized. Aheteroaryl group can be attached to the remainder of the molecule at anannular carbon or at an annular heteroatom. Heteroaryl may containadditional fused rings (e.g., from 1 to 3 rings), including additionallyfused aryl, heteroaryl, cycloalkyl, and/or heterocyclyl rings. Examplesof heteroaryl groups include, but are not limited to, pyridyl,pyrimidyl, thiophenyl, furanyl, thiazolyl, and the like.

“Heterocycle” or “heterocyclyl” refers to a saturated or an unsaturatednon-aromatic group having from 1 to 10 annular carbon atoms and from 1to 4 annular heteroatoms, such as nitrogen, sulfur or oxygen, and thelike, wherein the nitrogen and sulfur atoms are optionally oxidized, andthe nitrogen atom(s) are optionally quaternized. A heterocyclyl groupmay have a single ring or multiple condensed rings, but excludesheteroaryl groups. A heterocycle comprising more than one ring may befused, spiro or bridged, or any combination thereof. In fused ringsystems, one or more of the fused rings can be aryl or heteroaryl.Examples of heterocyclyl groups include, but are not limited to,tetrahydropyranyl, dihydropyranyl, piperidinyl, piperazinyl,pyrrolidinyl, thiazolinyl, thiazolidinyl, tetrahydrofuranyl,tetrahydrothiophenyl, 2,3-dihydrobenzo[b]thiophen-2-yl,4-amino-2-oxopyrimidin-1(2H)-yl, and the like.

“Oxo” refers to the moiety ═O.

“Optionally substituted” unless otherwise specified means that a groupmay be unsubstituted or substituted by one or more (e.g., 1, 2, 3, 4 or5) of the substituents listed for that group in which the substituentsmay be the same of different. In one embodiment, an optionallysubstituted group has one substituent. In another embodiment, anoptionally substituted group has two substituents. In anotherembodiment, an optionally substituted group has three substituents. Inanother embodiment, an optionally substituted group has foursubstituents. In some embodiments, an optionally substituted group has 1to 2, 2 to 5, 3 to 5, 2 to 3, 2 to 4, 3 to 4, 1 to 3, 1 to 4 or 1 to 5substituents.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results including clinical results. For example,beneficial or desired results include, but are not limited to, one ormore of the following: decreasing symptoms resulting from the disease,increasing the quality of life of those suffering from the disease,decreasing the dose of other medications required to treat the disease,delaying the progression of the disease, and/or prolonging survival ofindividuals. In reference to cancers or other unwanted cellproliferation, beneficial or desired results include shrinking a tumor(reducing tumor size); decreasing the growth rate of the tumor (such asto suppress tumor growth); reducing the number of cancer cells;inhibiting, retarding or slowing to some extent and preferably stoppingcancer cell infiltration into peripheral organs; inhibiting (slowing tosome extent and preferably stopping) tumor metastasis; inhibiting tumorgrowth; preventing or delaying occurrence and/or recurrence of tumor;and/or relieving to some extent one or more of the symptoms associatedwith the cancer. In some embodiments, beneficial or desired resultsinclude preventing or delaying occurrence and/or recurrence, such as ofunwanted cell proliferation.

As used herein, “delaying development of a disease” means to defer,hinder, slow, retard, stabilize, and/or postpone development of thedisease (such as cancer). This delay can be of varying lengths of time,depending on the history of the disease and/or individual being treated.As is evident to one skilled in the art, a sufficient or significantdelay can, in effect, encompass prevention, in that the individual doesnot develop the disease. For example, a late stage cancer, such asdevelopment of metastasis, may be delayed.

As used herein, an “effective dosage” or “effective amount” of compoundor salt thereof or pharmaceutical composition is an amount sufficient toeffect beneficial or desired results. For prophylactic use, beneficialor desired results include results such as eliminating or reducing therisk, lessening the severity of, or delaying the onset of the disease,including biochemical, histological and/or behavioral symptoms of thedisease, its complications and intermediate pathological phenotypespresenting during development of the disease. For therapeutic use,beneficial or desired results include ameliorating, palliating,lessening, delaying or decreasing one or more symptoms resulting fromthe disease, increasing the quality of life of those suffering from thedisease, decreasing the dose of other medications required to treat thedisease, enhancing effect of another medication such as via targeting,delaying the progression of the disease, and/or prolonging survival. Inreference to cancers or other unwanted cell proliferation, an effectiveamount comprises an amount sufficient to cause a tumor to shrink and/orto decrease the growth rate of the tumor (such as to suppress tumorgrowth) or to prevent or delay other unwanted cell proliferation. Insome embodiments, an effective amount is an amount sufficient to delaydevelopment. In some embodiments, an effective amount is an amountsufficient to prevent or delay occurrence and/or recurrence. Aneffective amount can be administered in one or more administrations, inthe case of cancer, the effective amount of the drug or composition may:(i) reduce the number of cancer cells; (ii) reduce tumor size; (iii)inhibit, retard, slow to some extent and preferably stop cancer cellinfiltration into peripheral organs; (iv) inhibit (i.e., slow to someextent and preferably stop) tumor metastasis; (v) inhibit tumor growth;(vi) prevent or delay occurrence and/or recurrence of tumor; and/or(vii) relieve to some extent one or more of the symptoms associated withthe cancer. An effective dosage can be administered in one or moreadministrations. For purposes of this disclosure, an effective dosage ofcompound or a salt thereof, or pharmaceutical composition is an amountsufficient to accomplish prophylactic or therapeutic treatment eitherdirectly or indirectly. It is intended and understood that an effectivedosage of a compound or salt thereof, or pharmaceutical composition mayor may not be achieved in conjunction with another drug, compound, orpharmaceutical composition. Thus, an “effective dosage” may beconsidered in the context of administering one or more therapeuticagents, and a single agent may be considered to be given in an effectiveamount if, in conjunction with one or more other agents, a desirableresult may be or is achieved.

As used herein, the term “individual” is a mammal, including humans. Anindividual includes, but is not limited to, human, bovine, horse,feline, canine, rodent, or primate. In some embodiments, the individualis human. The individual (such as a human) may have advanced disease orlesser extent of disease, such as low tumor burden. In some embodiments,the individual is at an early stage of a proliferative disease (such ascancer). In some embodiments, the individual is at an advanced stage ofa proliferative disease (such as an advanced cancer).

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

It is understood that aspects and variations described herein alsoinclude “consisting” and/or “consisting essentially of” aspects andvariations.

Compounds

In one aspect, provided is a compound of Formula (I):

or a salt thereof, wherein:Y is hydrogen or R⁴;m is 0, 1, 2, or 3;n is 0, 1, 2, 3, or 4;R¹ is independently F, Cl, or methyl;R² is C₁-C₆ alkyl, C₃-C₆ cycloalkyl or —(C₁-C₃ alkylene)CF₃;

R³ is

wherein:

indicates an aromatic ring;

M¹ is CH, CR^(3b) or N;

M² is CH, CR^(3b), N, or absent;

M³ is CH, CR^(3b), N, O, or S;

M⁴ is CH, CR^(3b), N, O, or S,

-   -   provided that:    -   (1) when M⁴ is O or S and M² is absent, then M³ is CH, CR^(3b)        or N, and    -   (2) when M³ is O or S and M² is absent, then M⁴ is CH, CR^(3b)        or N;

R^(3a) is C₃-C₆ cycloalkyl optionally substituted by C₁-C₆ haloalkyl or—CN, or C₁-C₆ alkyl optionally substituted by halogen, —OH or —CN,provided that when R^(3a) is C₁-C₆ alkyl optionally substituted byhalogen, —OH or —CN, then at least one of M¹, M², M³, and M⁴ is CR³b;

R^(3b) is halogen or —CN;

each R⁴ is independently oxo, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,halogen, —C(O)R¹⁷, —C(O)OR¹⁷, —C(O)NR¹⁷R¹⁸, —CN, —Si(C₁-C₆ alkyl)₃,—OR¹⁷, —NR¹⁷R¹⁸, —OC(O)NR¹⁷R¹⁸, —NR¹⁷C(O)R¹⁸, —S(O)₂R¹⁷, —NR¹⁷S(O)₂R¹⁸,—S(O)₂NR¹⁷R¹⁸, C₃-C₆ cycloalkyl, 3- to 6-membered heterocyclyl, —(C₁-C₃alkylene)CN, —(C₁-C₃ alkylene)OR¹⁷, —(C₁-C₃ alkylene)NR¹⁷R¹⁸, —(C₁-C₃alkylene)CF₃, —(C₁-C₃ alkylene)C(O)R¹⁷, —(C₁-C₃ alkylene)C(O)NR¹⁷R¹⁸,—(C₁-C₃ alkylene)NR¹⁷C(O)R¹⁸, —(C₁-C₃ alkylene)S(O)₂R¹⁷, —(C₁-C₃alkylene)NR¹⁷S(O)₂R¹⁸, —(C₁-C₃ alkylene)S(O)₂NR¹⁷R¹⁸, —(C₁-C₃alkylene)(C₃-C₆ cycloalkyl) or —(C₁-C₃ alkylene)(3- to 6-memberedheterocyclyl), wherein each R⁴ is independently optionally substitutedby halogen, oxo, —OR¹⁹, —NR¹⁹R²⁰, or —C(O)R¹⁹ or two R⁴, when bound tothe same carbon are taken together with the carbon to which they areattached to form a C₃-C₆ cycloalkyl or 3- to 6-membered heterocyclyl,each is optionally substituted by R¹⁹;each R¹⁷, R¹⁸, R¹⁹, and R²⁰ is independently hydrogen, C₃-C₆ cycloalkyl,3-6 membered heterocyclyl or C₁-C₆ alkyl, each of which is optionallysubstituted by halogen, oxo or —OH,

or R¹⁷ and R¹⁸ are taken together with the atom to which they attachedto form a 3-6 membered heterocyclyl optionally substituted by halogen,oxo or —OH.

In some embodiments of a compound of Formula (I), or a salt thereof, thecompound is other than the compounds in Table 1X or a salt thereof. Insome embodiments of a compound of Formula (I), or a salt thereof, thecompound is other than the Compound Nos. 1x-39x in Table 1X or a saltthereof.

TABLE 1X  1x 1-(6-Cyclopropylpyridin-2-yl)-2-ethyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one  2x1-(6-Cyclopropylpyridin-2-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one  3x1-(6-Cyclopropylpyridin-2-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one  4x1-(3-(Tert-butyl)-4-fluorophenyl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one  5x1-(3-Cyclopropyl-4-fluorophenyl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one  6x1-(3-(Tert-butyl)-2-fluorophenyl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one  7x1-(3-Cyclopropyl-2-fluorophenyl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one  8x2-(Tert-butyl)-4-(2-isopropyl-3-oxo-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)benzonitrile  9x2-(2-Hydroxypropan-2-yl)-4-(2-isopropyl-3-oxo-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)benzonitrile 10x2-Cyclopropyl-4-(2-isopropyl-3-oxo-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)benzonitrile 11x2-Cyclopropyl-6-(2-isopropyl-3-oxo-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)benzonitrile 12x2-(Tert-butyl)-6-(2-isopropyl-3-oxo-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)benzonitrile 13x1-(3-(Tert-butyl)-4-chloro-2-fluorophenyl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 14x1-(3-(Tert-butyl)-2,4-difluorophenyl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 15x1-(2-(Tert-butyl)-3-fluoropyridin-4-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 16x1-(6-(Tert-butyl)-5-fluoropyridin-2-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 17x1-(6-(Tert-butyl)-5-fluoropyridin-2-yl)-2-isopropyl-6-((7-methyl-1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 18x1-(2-Cyclopropylpyridin-4-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 19x2-Isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1-(2-(1-(trifluoromethyl)cyclopropyl)pyridin-4-yl)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 20x1-(3-(Tert-butyl)-4-fluorophenyl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 21x1-(3-Cyclopropyl-4-fluorophenyl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 22x1-(3-(Tert-butyl)-2-fluorophenyl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 23x1-(3-Cyclopropyl-2-fluorophenyl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 24x2-(Tert-butyl)-4-(2-isopropyl-3-oxo-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)benzonitrile 25x2-(2-Hydroxypropan-2-yl)-4-(2-isopropyl-3-oxo-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)benzonitrile 26x2-Cyclopropyl-4-(2-isopropyl-3-oxo-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)benzonitrile 27x2-Cyclopropyl-6-(2-isopropyl-3-oxo-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)benzonitrile 28x2-(Tert-butyl)-6-(2-isopropyl-3-oxo-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)benzonitrile 29x1-(3-(Tert-butyl)-4-chloro-2-fluorophenyl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 30x1-(3-(Tert-butyl)-2,4-difluorophenyl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 31x1-(2-(Tert-butyl)-3-fluoropyridin-4-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 32x1-(6-(Tert-butyl)-5-fluoropyridin-2-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 33x1-(6-(Tert-butyl)-5-fluoropyridin-2-yl)-2-isopropyl-6-((6-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 34x1-(2-Cyclopropylpyridin-4-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 35x2-Isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1-(2-(1-(trifluoromethyl)cyclopropyl)pyridin-4-yl)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 36x1-(2-(Tert-butyl)-5-fluoropyridin-4-yl)-2-ethyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 37x1-(6-(Tert-butyl)-3-fluoropyridin-2-yl)-2-ethyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 38x2-Ethyl-1-(3-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 39x1-(2-(Tert-butyl)-5-fluoropyridin-4-yl)-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-2-(2,2,2-trifluoroethyl)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 40x1-(4-Fluoro-3-(2-hydroxypropan-2-yl)phenyl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 41x1-(4-Fluoro-3-(2-hydroxypropan-2-yl)phenyl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one 42x1-(2-(Tert-butyl)-5-fluoropyridin-4-yl)-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-2-(2,2,2-trifluoroethyl)-1,2-dihydro-3H-pyrazolo[3,4- d]pyrimidin-3-one

In some embodiments of a compound of Formula (I), the compound is ofFormula (II):

In some embodiments of a compound of Formula (I), the compound is ofFormula (III):

In some embodiments of a compound of Formula (I), R² is C₁-C₆ alkyl,such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,or sec-butyl. In some embodiments, R² is isopropyl or ethyl. In someembodiments, R² is isopropyl. In some embodiments, R² is ethyl. In someembodiments, R² is C₃₋₆ cycloalkyl, such as cyclopropyl, cyclobutyl,cyclopentyl, or cyclohexyl. In some embodiments, R² is cyclopropyl. Insome embodiments, R² is —(C₁-C₃ alkylene)CF₃. In some embodiments, R² is—CH₂CF₃. In some embodiments, R² is selected from the group consistingof isopropyl, ethyl, cyclopropyl, and —CH₂CF₃.

In some embodiments of a compound of Formula (I), M¹ is CH. In someembodiments, M¹ is CR^(3b). In some embodiments, M¹ is N.

In some embodiments of a compound of Formula (I), M² is CH. In someembodiments, M² is CR^(3b). In some embodiments, M² is N. In someembodiments, M² is absent.

In some embodiments of a compound of Formula (I), M³ is CH. In someembodiments, M³ is CR^(3b). In some embodiments, M³ is N. In someembodiments, M³ is S. In some embodiments, M³ is O.

In some embodiments of a compound of Formula (I), M⁴ is CH. In someembodiments, M⁴ is CR^(3b). In some embodiments, M⁴ is N. In someembodiments, M⁴ is S. In some embodiments, M⁴ is O. In some embodiments,when M⁴ is O or S and M² is absent, then M³ is CH, CR^(3b) or N. In someembodiments, when M³ is O or S and M² is absent, then M⁴ is CH, CR^(3b)or N.

In some embodiments of a compound of Formula (I), R³ is selected fromthe group consisting of:

In some embodiments, R³ is

In some embodiments, R³ is

In some embodiments, R³ is

In some embodiments, R³ is

In some embodiments, R³ is

In some embodiments, R³ is

In some embodiments, R³ is

In some embodiments, R³ is

In some embodiments, R³ is

In some embodiments, R³ is

In some embodiments, R³ is

In some embodiments, R³ is

In some embodiments, R³ is

In some embodiments, R³ is

In some embodiments of a compound of Formula (I), R^(3a) is C₃-C₆cycloalkyl optionally substituted by C₁-C₆ haloalkyl or —CN, such ascyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, each of which isoptionally substituted by C₁-C₆ haloalkyl or —CN. In some embodiments,R^(3a) is C₃₋₆ cycloalkyl which is unsubstituted, such as cyclopropyl,cyclobutyl, cyclopentyl, or cyclohexyl, each of which is unsubstituted.In some embodiments, R^(3a) is C₃-C₆ cycloalkyl optionally substitutedby C₁-C₆ haloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, orcyclohexyl, each of which is optionally substituted by C₁-C₆ haloalkyl.In some embodiments, R^(3a) is C₃-C₆ cycloalkyl optionally substitutedby —CN, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl,each of which is optionally substituted by —CN. In some embodiments,R^(3a) is

In some embodiments, R^(3a) is

In some embodiments, R^(3a) is

In some embodiments, R^(3a) is

In some embodiments, R^(3a) is

In some embodiments, R^(3a) is C₁-C₆ alkyl optionally substituted byhalogen, —OH or —CN, such as methyl, ethyl, n-propyl, isopropyl,n-butyl, t-butyl, isobutyl, or sec-butyl, each of which is optionallysubstituted by halogen, —OH or —CN. In some embodiments, R^(3a) is C₁-C₆alkyl which is unsubstituted, such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, or sec-butyl, each of which isunsubstituted. In some embodiments, R^(3a) is C₁-C₆ alkyl optionallysubstituted by halogen, such as methyl, ethyl, n-propyl, isopropyl,n-butyl, t-butyl, isobutyl, or sec-butyl, each of which is optionallysubstituted by halogen. In some embodiments, R^(3a) is C₁-C₆ alkyloptionally substituted by —OH, such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, or sec-butyl, each of which isoptionally substituted by —OH. In some embodiments, R^(3a) is C₁-C₆alkyl optionally substituted by —OH, such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, or sec-butyl, each of which isoptionally substituted by —CN. In some embodiments, R^(3a) is

In some embodiments, R^(3a) is

In some embodiments, R^(3a) is

In some embodiments, R^(3a) is

In some embodiments, R^(3a) is

In some embodiments, R^(3a) is

In some embodiments, R^(3a) is

In some embodiments, R^(3a) is

In some embodiments of a compound of Formula (I), R^(3b) is —CN. In someembodiments, R^(3b) is halogen, such as fluoro, chloro, bromo, or iodo.In some embodiments, R^(3b) is fluoro. In some embodiments, R^(3b) ischloro. In some embodiments, R^(3b) is bromo.

In some embodiments of a compound of Formula (I), R³ is selected fromthe group consisting of:

In some embodiments of a compound of Formula (I), m is 0. In someembodiments, m is 1. In some embodiments, m is 2. In some embodiments, mis 3. In some embodiments, m is 0, 1, or 2. In some embodiments, m is 0or 1.

In some embodiments of a compound of Formula (I), R¹ is F. In someembodiments, R¹ is C₁. In some embodiments R¹ is methyl.

In some embodiments of a compound of Formula (I), n is 0. In someembodiments, n is 1. In some embodiments, n is 2. In some embodiments, nis 3. In some embodiments, n is 4. In some embodiments, n is 0, 1, 2, or3. In some embodiments, n is 0, 1, or 2. In some embodiments, n is 0 or1.

In some embodiments of a compound of Formula (I), each R⁴ isindependently C₁-C₆ alkyl, or two R⁴, when bound to the same carbon, aretaken together with the carbon to which they are attached to form aC₃-C₆ cycloalkyl. In some embodiments, each R⁴ is independently C₁-C₆alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl,isobutyl, or sec-butyl. In some embodiments, n is 1 and R⁴ is C₁-C₆alkyl. In some embodiments, n is 2 and each R⁴ is independently C₁-C₆alkyl. In some embodiments, n is 2 and each R⁴ is methyl. In someembodiments, n is 2 and two R⁴, when bound to the same carbon, are takentogether with the carbon to which they are attached to form a C₃-C₆cycloalkyl.

In some embodiments of a compound of Formula (I), Y is hydrogen. In someembodiments, Y is R⁴. In some embodiments, Y is C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, or 3- to 6-memberedheterocyclyl. In some embodiments, Y is C₁-C₆ alkyl, such as methyl,ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, or sec-butyl.

In some embodiments of a compound of Formula (I), ring A, ring B, Y, R¹and R⁴ together are taken together to form a moiety selected from thegroup consisting of:

In some embodiments, ring A, ring B, Y, R¹ and R⁴ together are takentogether to form a moiety selected from the group consisting of:

In some embodiments, ring A, ring B, Y, R¹ and R⁴ together are takentogether to form

In some embodiments, ring A, ring B, Y, R¹ and R⁴ together are takentogether to form

In some embodiments, ring A, ring B, Y, R¹ and R⁴ together are takentogether to form

In some embodiments, ring A, ring B, Y, R¹ and R⁴ together are takentogether to form

In some embodiments, ring A, ring B, Y, R¹ and R⁴ together are takentogether to form

In some embodiments, ring A, ring B, Y, R¹ and R⁴ together are takentogether to form

In some embodiments of a compound of Formula (I), the compound has oneor more of the following features:

(I) R² is

(1) C₁-C₆ alkyl, such as isopropyl or ethyl,

(2) C₃-C₆ cycloalkyl, such as cyclopropyl, or

(3) —(C₁-C₃ alkylene)CF₃, such as —CH₂CF₃;

(II) R³ is

(4)

wherein R^(3a) is C₃-C₆ cycloalkyl optionally substituted by C₁-C₆haloalkyl or —CN, or C₁-C₆ alkyl optionally substituted by halogen, —OHor —CN, provided that when R^(3a) is C₁-C₆ alkyl optionally substitutedby halogen, —OH or —CN, then at least one of M¹, M², M³, and M⁴ isCR^(3b), and R^(3b) is halogen or —CN, or(5)

(III) ring A, ring B, R¹, and R⁴ are taken together to form a moietyselected from the group consisting of:

In some embodiments, (1) applies. In some embodiments, (2) applies. Insome embodiments, (3) applies. In some embodiments, (4) applies. In someembodiments, (5) applies. In some embodiments, (III) applies. In someembodiments, (I) and (4) apply. In some embodiments, (I) and (5) apply.In some embodiments, (1) and (4) apply. In some embodiments, (1) and (5)apply. In some embodiments, (2) and (4) apply. In some embodiments, (2)and (5) apply. In some embodiments, (3) and (4) apply. In someembodiments, (3) and (5) apply. In some embodiments, (I) and (III)apply. In some embodiments, (1) and (III) apply. In some embodiments,(2) and (III) apply. In some embodiments, (3) and (III) apply. In someembodiments, (4) and (III) apply. In some embodiments, (5) and (III)apply. In some embodiments, (I), (4), and (III) apply. In someembodiments, (I), (5), and (III) apply. In some embodiments, (1), (4),and (III) apply. In some embodiments, (1), (5), and (III) apply. In someembodiments, (2), (4), and (III) apply. In some embodiments, (2), (5),and (III) apply. In some embodiments, (3), (4), and (III) apply. In someembodiments, (3), (5), and (III) apply.

In the descriptions herein, it is understood that every description,variation, embodiment or aspect of a moiety may be combined with everydescription, variation, embodiment or aspect of other moieties the sameas if each and every combination of descriptions is specifically andindividually listed. For example, every description, variation,embodiment or aspect provided herein with respect to R¹ of Formula (I)may be combined with every description, variation, embodiment or aspectof R², R³, R⁴, m, n, and Y the same as if each and every combinationwere specifically and individually listed. It is also understood thatall descriptions, variations, embodiments or aspects of Formula (I),where applicable, apply equally to other formulae detailed herein, andare equally described, the same as if each and every description,variation, embodiment or aspect were separately and individually listedfor all formulae. For example, all descriptions, variations, embodimentsor aspects of formula (I), where applicable, apply equally to any offormulae as detailed herein, such as Formula (II) and Formula (III) andare equally described, the same as if each and every description,variation, embodiment or aspect were separately and individually listedfor all formulae.

Also provided are salts of compounds referred to herein, such aspharmaceutically acceptable salts. The invention also includes any orall of the stereochemical forms, including any enantiomeric ordiastereomeric forms, and any tautomers or other forms of the compoundsdescribed.

A compound as detailed herein may in one aspect be in a purified formand compositions comprising a compound in purified forms are detailedherein. Compositions comprising a compound as detailed herein or a saltthereof are provided, such as compositions of substantially purecompounds. In some embodiments, a composition containing a compound asdetailed herein or a salt thereof is in substantially pure form. Unlessotherwise stated, “substantially pure” intends a composition thatcontains no more than 35% impurity, wherein the impurity denotes acompound other than the compound comprising the majority of thecomposition or a salt thereof. In some embodiments, a composition ofsubstantially pure compound or a salt thereof is provided wherein thecomposition contains no more than 25%, 20%, 15%, 10%, or 5% impurity. Insome embodiments, a composition of substantially pure compound or a saltthereof is provided wherein the composition contains or no more than 3%,2%, 1% or 0.5% impurity.

Representative compounds are listed in Table 1.

TABLE 1 Compound No. Structure 1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

1.10

1.11

1.12

1.13

1.14

1.15

1.16

1.17

1.18

1.19

1.20

1.21

1.22

1.23

1.24

1.25

1.26

1.27

1.28

1.29

1.30

1.31

1.32

1.33

1.34

1.35

1.36

1.37

1.38

1.39

1.40

1.41

1.42

1.43

1.44

1.45

1.46

1.47

1.48

1.49

1.50

1.51

1.52

1.53

1.54

1.55

1.56

1.57

1.58

1.59

1.60

1.61

1.62

1.63

1.64

1.65

1.66

1.67

1.68

1.69

1.70

1.71

1.72

1.73

1.74

1.75

1.76

1.77

1.78

1.79

1.80

1.81

1.82

1.83

1.84

1.85

1.86

1.87

1.88

1.89

1.90

1.91

1.92

1.93

1.94

1.95

1.96

1.97

1.98

1.99

1.100

1.101

1.102

1.103

1.104

1.105

1.106

1.107

1.108

1.109

1.110

1.111

1.112

1.113

1.114

1.115

1.116

1.117

1.118

1.119

1.120

1.121

1.122

1.123

1.124

1.125

1.126

1.127

1.128

1.129

1.130

1.131

1.132

1.133

1.134

1.135

1.136

1.137

1.138

1.139

1.140

1.141

1.142

1.143

1.144

1.145

1.146

1.147

1.148

1.149

1.150

1.151

1.152

1.153

1.154

1.155

1.156

1.157

1.158

1.159

1.160

1.161

1.162

1.163

1.164

1.165

1.166

1.167

1.168

1.169

1.170

1.171

1.172

1.173

1.174

1.175

1.176

1.177

1.178

1.179

1.180

1.181

1.182

1.183

1.184

1.185

1.186

1.187

1.188

1.189

1.190

1.191

1.192

1.193

1.194

1.195

1.196

1.197

1.198

1.199

1.200

1.201

1.202

1.203

1.204

1.205

1.206

1.207

1.208

1.209

1.210

1.211

1.212

1.213

1.214

1.215

1.216

1.217

1.218

1.219

1.220

1.221

1.222

1.223

1.224

1.225

1.226

1.227

1.228

1.229

1.230

1.231

1.232

1.233

1.234

1.235

1.236

1.237

1.238

1.239

1.240

1.241

1.242

1.243

1.244

1.245

1.246

1.247

1.248

1.249

1.250

1.251

1.252

1.253

1.254

1.255

1.256

1.257

1.258

1.259

1.260

1.261

1.262

1.263

1.264

1.265

1.266

1.267

1.268

1.269

1.270

1.271

1.272

1.273

1.274

1.275

1.276

1.277

1.278

1.279

1.280

1.281

1.282

1.283

1.284

1.285

1.286

1.287

1.288

1.289

1.290

1.291

1.292

1.293

1.294

1.295

1.296

1.297

1.298

1.299

1.300

1.301

1.302

1.303

1.304

1.305

1.306

1.307

1.308

1.309

1.310

1.311

1.312

1.313

1.314

1.315

1.316

1.317

1.318

1.319

1.320

1.321

1.322

1.323

1.324

1.325

1.326

1.327

1.328

1.329

1.330

1.331

1.332

1.333

1.334

1.335

1.336

1.337

1.338

1.339

1.340

1.341

1.342

1.343

1.344

1.345

1.346

1.347

1.348

1.349

1.350

1.351

1.352

1.353

1.354

1.355

1.356

1.357

1.358

1.359

1.360

1.361

1.362

1.363

1.364

1.365

1.366

1.367

1.368

1.369

1.370

1.371

1.372

1.373

1.374

1.375

1.376

1.377

1.378

1.379

1.380

1.381

1.382

1.383

1.384

1.385

1.386

1.387

1.388

1.389

1.390

1.391

1.392

1.393

1.394

1.395

1.396

1.397

1.398

1.399

1.400

1.401

1.402

1.403

1.404

1.405

1.406

1.407

1.408

1.409

1.410

1.411

1.412

1.413

1.414

1.415

1.416

1.417

1.418

1.419

1.420

1.421

1.422

1.423

1.424

1.425

1.426

1.427

1.428

1.429

1.430

1.431

1.432

1.433

1.434

1.435

1.436

1.437

1.438

1.439

1.440

1.441

1.442

1.443

1.444

1.445

1.446

1.447

1.448

1.449

1.450

1.451

1.452

1.453

1.454

1.455

1.456

1.457

1.458

1.459

1.460

1.461

1.462

1.463

1.464

1.465

1.466

1.467

1.468

1.469

1.470

1.471

1.472

1.473

1.474

1.475

1.476

1.477

1.478

1.479

1.480

1.481

1.482

1.483

1.484

1.485

1.486

1.487

1.488

1.489

In some embodiments, provided herein is a compound described in Table 1,or a tautomer thereof, or a salt of any of the foregoing, and usesthereof. In some embodiments, provided herein is a compound described inTable 1 or a pharmaceutically acceptable salt thereof.

The embodiments and variations described herein are suitable forcompounds of any formulae detailed herein, where applicable.

Representative examples of compounds detailed herein, includingintermediates and final compounds according to the present disclosureare depicted herein. It is understood that in one aspect, any of thecompounds may be used in the methods detailed herein, including, whereapplicable, intermediate compounds that may be isolated and administeredto an individual.

The compounds depicted herein may be present as salts even if salts arenot depicted and it is understood that the present disclosure embracesall salts and solvates of the compounds depicted here, as well as thenon-salt and non-solvate form of the compound, as is well understood bythe skilled artisan. In some embodiments, the salts of the compoundsprovided herein are pharmaceutically acceptable salts. Where one or moretertiary amine moiety is present in the compound, the N-oxides are alsoprovided and described.

Where tautomeric forms may be present for any of the compounds describedherein, each and every tautomeric form is intended even though only oneor some of the tautomeric forms may be explicitly depicted. Thetautomeric forms specifically depicted may or may not be the predominantforms in solution or when used according to the methods describedherein.

The present disclosure also includes any or all of the stereochemicalforms, including any enantiomeric or diastereomeric forms of thecompounds described, such as the compounds of Table 1. The structure orname is intended to embrace all possible stereoisomers of a compounddepicted, and each unique stereoisomer has a compound number bearing asuffix “a”, “b”, etc. All forms of the compounds are also embraced bythe invention, such as crystalline or non-crystalline forms of thecompounds. Compositions comprising a compound of the invention are alsointended, such as a composition of substantially pure compound,including a specific stereochemical form thereof, or a compositioncomprising mixtures of compounds of the invention in any ratio,including two or more stereochemical forms, such as in a racemic ornon-racemic mixture.

The invention also intends isotopically-labeled and/orisotopically-enriched forms of compounds described herein. The compoundsherein may contain unnatural proportions of atomic isotopes at one ormore of the atoms that constitute such compounds. In some embodiments,the compound is isotopically-labeled, such as an isotopically-labeledcompound of Formula (I) or variations thereof described herein, where afraction of one or more atoms are replaced by an isotope of the sameelement. Exemplary isotopes that can be incorporated into compounds ofthe invention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorus, sulfur, chlorine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C ¹³N, ¹⁵O,¹⁷O, ³²P, ³⁵S, ¹⁸F, ³⁶C₁. Certain isotope labeled compounds (e.g. ³H and¹⁴C) are useful in compound or substrate tissue distribution studies.Incorporation of heavier isotopes such as deuterium (²H) can affordcertain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life, or reduced dosagerequirements and, hence may be preferred in some instances.

Isotopically-labeled compounds of the present invention can generally beprepared by standard methods and techniques known to those skilled inthe art or by procedures similar to those described in the accompanyingExamples substituting appropriate isotopically-labeled reagents in placeof the corresponding non-labeled reagent.

The invention also includes any or all metabolites of any of thecompounds described. The metabolites may include any chemical speciesgenerated by a biotransformation of any of the compounds described, suchas intermediates and products of metabolism of the compound, such aswould be generated in vivo following administration to a human.

Articles of manufacture comprising a compound described herein, or asalt or solvate thereof, in a suitable container are provided. Thecontainer may be a vial, jar, ampoule, preloaded syringe, i.v. bag, andthe like.

Preferably, the compounds detailed herein are orally bioavailable.However, the compounds may also be formulated for parenteral (e.g.,intravenous) administration.

One or several compounds described herein can be used in the preparationof a medicament by combining the compound or compounds as an activeingredient with a pharmacologically acceptable carrier, which are knownin the art. Depending on the therapeutic form of the medication, thecarrier may be in various forms. In one variation, the manufacture of amedicament is for use in any of the methods disclosed herein, e.g., forthe treatment of cancer.

General Synthetic Methods

The compounds of the invention may be prepared by a number of processesas generally described below and more specifically in the Exampleshereinafter (such as the schemes provided in the Examples below). In thefollowing process descriptions, the symbols when used in the formulaedepicted are to be understood to represent those groups described abovein relation to the formulae herein.

Where it is desired to obtain a particular enantiomer of a compound,this may be accomplished from a corresponding mixture of enantiomersusing any suitable conventional procedure for separating or resolvingenantiomers. Thus, for example, diastereomeric derivatives may beproduced by reaction of a mixture of enantiomers, e.g., a racemate, andan appropriate chiral compound. The diastereomers may then be separatedby any convenient means, for example by crystallization and the desiredenantiomer recovered. In another resolution process, a racemate may beseparated using chiral High Performance Liquid Chromatography.Alternatively, if desired a particular enantiomer may be obtained byusing an appropriate chiral intermediate in one of the processesdescribed.

Chromatography, recrystallization and other conventional separationprocedures may also be used with intermediates or final products whereit is desired to obtain a particular isomer of a compound or tootherwise purify a product of a reaction.

Solvates and/or polymorphs of a compound provided herein or a saltthereof are also contemplated. Solvates contain either stoichiometric ornon-stoichiometric amounts of a solvent, and are often formed during theprocess of crystallization. Hydrates are formed when the solvent iswater, or alcoholates are formed when the solvent is alcohol. Polymorphsinclude the different crystal packing arrangements of the same elementalcomposition of a compound. Polymorphs usually have different X-raydiffraction patterns, infrared spectra, melting points, density,hardness, crystal shape, optical and electrical properties, stability,and/or solubility. Various factors such as the recrystallizationsolvent, rate of crystallization, and storage temperature may cause asingle crystal form to dominate

In some embodiments, compounds of Formula (I), (II) or (III) aresynthesized according to Scheme 1 to Scheme 5.

wherein m, n, Y, R¹, R², R³, and R⁴ are as defined herein for Formula(I). Particular examples are provided in the Example Section below.

Pharmaceutical Compositions and Formulations

Pharmaceutical compositions of any of the compounds detailed herein areembraced by this disclosure. Thus, the present disclosure includespharmaceutical compositions comprising a compound as detailed herein ora salt thereof and a pharmaceutically acceptable carrier or excipient.In one aspect, the pharmaceutically acceptable salt is an acid additionsalt, such as a salt formed with an inorganic or organic acid.Pharmaceutical compositions may take a form suitable for oral, buccal,parenteral, nasal, topical or rectal administration or a form suitablefor administration by inhalation.

A compound as detailed herein may in one aspect be in a purified formand compositions comprising a compound in purified forms are detailedherein. Compositions comprising a compound as detailed herein or a saltthereof are provided, such as compositions of substantially purecompounds. In some embodiments, a composition containing a compound asdetailed herein or a salt thereof is in substantially pure form.

In one variation, the compounds herein are synthetic compounds preparedfor administration to an individual. In another variation, compositionsare provided containing a compound in substantially pure form. Inanother variation, the present disclosure embraces pharmaceuticalcompositions comprising a compound detailed herein and apharmaceutically acceptable carrier. In another variation, methods ofadministering a compound are provided. The purified forms,pharmaceutical compositions and methods of administering the compoundsare suitable for any compound or form thereof detailed herein.

A compound detailed herein or salt thereof may be formulated for anyavailable delivery route, including an oral, mucosal (e.g., nasal,sublingual, vaginal, buccal or rectal), parenteral (e.g., intramuscular,subcutaneous or intravenous), topical or transdermal delivery form. Acompound or salt thereof may be formulated with suitable carriers toprovide delivery forms that include, but are not limited to, tablets,caplets, capsules (such as hard gelatin capsules or soft elastic gelatincapsules), cachets, troches, lozenges, gums, dispersions, suppositories,ointments, cataplasms (poultices), pastes, powders, dressings, creams,solutions, patches, aerosols (e.g., nasal spray or inhalers), gels,suspensions (e.g., aqueous or non-aqueous liquid suspensions,oil-in-water emulsions or water-in-oil liquid emulsions), solutions andelixirs.

One or several compounds described herein or a salt thereof can be usedin the preparation of a formulation, such as a pharmaceuticalformulation, by combining the compound or compounds, or a salt thereof,as an active ingredient with a pharmaceutically acceptable carrier, suchas those mentioned above. Depending on the therapeutic form of thesystem (e.g., transdermal patch vs. oral tablet), the carrier may be invarious forms. In addition, pharmaceutical formulations may containpreservatives, solubilizers, stabilizers, re-wetting agents, emulgators,sweeteners, dyes, adjusters, and salts for the adjustment of osmoticpressure, buffers, coating agents or antioxidants. Formulationscomprising the compound may also contain other substances which havevaluable therapeutic properties. Pharmaceutical formulations may beprepared by known pharmaceutical methods. Suitable formulations can befound, e.g., in Remington's Pharmaceutical Sciences, Mack PublishingCompany, Philadelphia, Pa., 20^(th) ed. (2000), which is incorporatedherein by reference.

Compounds as described herein may be administered to individuals in aform of generally accepted oral compositions, such as tablets, coatedtablets, and gel capsules in a hard or in soft shell, emulsions orsuspensions. Examples of carriers, which may be used for the preparationof such compositions, are lactose, corn starch or its derivatives, talc,stearate or its salts, etc. Acceptable carriers for gel capsules withsoft shell are, for instance, plant oils, wax, fats, semisolid andliquid poly-ols, and so on. In addition, pharmaceutical formulations maycontain preservatives, solubilizers, stabilizers, re-wetting agents,emulgators, sweeteners, dyes, adjusters, and salts for the adjustment ofosmotic pressure, buffers, coating agents or antioxidants.

Any of the compounds described herein can be formulated in a tablet inany dosage form described, for example, a compound as described hereinor a salt thereof can be formulated as a 10 mg tablet.

Compositions comprising a compound provided herein are also described.In one variation, the composition comprises a compound or salt thereofand a pharmaceutically acceptable carrier or excipient. In anothervariation, a composition of substantially pure compound is provided.

Methods of Use

Compounds and compositions detailed herein, such as a pharmaceuticalcomposition containing a compound of any formula provided herein or asalt thereof and a pharmaceutically acceptable carrier or excipient, maybe used in methods of administration and treatment as provided herein.The compounds and compositions may also be used in in vitro methods,such as in vitro methods of administering a compound or composition tocells for screening purposes and/or for conducting quality controlassays.

Provided herein is a method of treating a disease in an individualcomprising administering an effective amount of a compound of Formula(I), (II) or (III) or any embodiment, variation or aspect thereof(collectively, a compound of Formula (I), (II) or (III) or the presentcompounds or the compounds detailed or described herein) or apharmaceutically acceptable salt thereof, to the individual. Furtherprovided herein is a method of treating a proliferative disease in anindividual, comprising administering an effective amount of a compoundof Formula (I), (II) or (III) or a pharmaceutically acceptable saltthereof, to the individual. Also provided herein is a method of treatingcancer in an individual comprising administering an effective amount ofa compound of Formula (I), (II) or (III) or a pharmaceuticallyacceptable salt thereof, to the individual. In some embodiments, thecompound is administered to the individual according to a dosage and/ormethod of administration described herein.

In some embodiments, the cancer in the individual has one or more TP53gene mutations or expresses mutant p53. In some embodiments, the cancerin the individual that has one or more TP53 gene mutations or expressesmutant p53 is glioblastoma. TP53 is the human gene that encodes p53. Insome embodiments, provided herein is a method of treating a cancer in anindividual, comprising (a) selecting the individual for treatment basedon (i) the presence of one or more mutations of the TP53 gene in thecancer, or (ii) expression of mutant p53 in the cancer, andadministering an effective amount of a compound of Formula (I), (II) or(III) or a pharmaceutically acceptable salt thereof, to the individual.In some embodiments, the cancer is assayed for the expression of mutantp53. In some embodiments, the TP53 gene of the cancer is sequenced todetect the one or more mutations. In some embodiments, the TP53 gene issequenced by biopsying the cancer and sequencing the TP53 gene from thebiopsied cancer. In some embodiments, the TP53 gene is sequenced bysequencing circulating-tumor DNA (ctDNA) from the individual.

In some embodiments, provided herein is a method of using a compound ofFormula (I), (II) or (III) or any embodiment in the manufacture of amedicament for treatment of a disease. In some embodiments, providedherein is a method of using a compound of Formula (I), (II) or (III) orany embodiment in the manufacture of a medicament for treatment ofcancer.

In some embodiments, a compound of Formula (I), (II) or (III) or a saltthereof is used to treat an individual having a proliferative disease,such as cancer as described herein. In some embodiments, the individualis at risk of developing a proliferative disease, such as cancer. Insome of these embodiments, the individual is determined to be at risk ofdeveloping cancer based upon one or more risk factors. In some of theseembodiments, the risk factor is a family history and/or gene associatedwith cancer.

The present compounds or salts thereof are believed to be effective fortreating a variety of diseases and disorders. For example, in someembodiments, the present compositions may be used to treat aproliferative disease, such as cancer. In some embodiments the cancer isa solid tumor. In some embodiments the cancer is any of adult andpediatric oncology, myxoid and round cell carcinoma, locally advancedtumors, metastatic cancer, human soft tissue sarcomas, including Ewing'ssarcoma, cancer metastases, including lymphatic metastases, squamouscell carcinoma, particularly of the head and neck, esophageal squamouscell carcinoma, oral carcinoma, blood cell malignancies, includingmultiple myeloma, leukemias, including acute lymphocytic leukemia, acutenonlymphocytic leukemia, chronic lymphocytic leukemia, chronicmyelocytic leukemia, and hairy cell leukemia, effusion lymphomas (bodycavity based lymphomas), thymic lymphoma, cutaneous T cell lymphoma,Hodgkin's lymphoma, non-Hodgkin's lymphoma, cancer of the adrenalcortex, ACTH-producing tumors, lung cancer, including small cellcarcinoma and nonsmall cell cancers, breast cancer, including small cellcarcinoma and ductal carcinoma, gastrointestinal cancers, includingstomach cancer, colon cancer, colorectal cancer, polyps associated withcolorectal neoplasia, pancreatic cancer, liver cancer, urologicalcancers, including bladder cancer, including primary superficial bladdertumors, invasive transitional cell carcinoma of the bladder, andmuscle-invasive bladder cancer, prostate cancer, malignancies of thefemale genital tract, including ovarian carcinoma, primary peritonealepithelial neoplasms, cervical carcinoma, uterine endometrial cancers,vaginal cancer, cancer of the vulva, uterine cancer and solid tumors inthe ovarian follicle, malignancies of the male genital tract, includingtesticular cancer and penile cancer, kidney cancer, including renal cellcarcinoma, brain cancer, including intrinsic brain tumors,neuroblastoma, astrocytic brain tumors, gliomas, glioblastoma,metastatic tumor cell invasion in the central nervous system, bonecancers, including osteomas and osteosarcomas, skin cancers, includingmelanoma, tumor progression of human skin keratinocytes, squamous cellcancer, thyroid cancer, retinoblastoma, neuroblastoma, peritonealeffusion, malignant pleural effusion, mesothelioma, Wilms's tumors, gallbladder cancer, trophoblastic neoplasms, hemangiopericytoma, andKaposi's sarcoma.

In some embodiments, the compounds and compositions described hereinsuppress G₂-M checkpoint in a cell (such as a cancer cell). In someembodiments, the cancer cell is a cancer cell from any of the cancertypes described herein. Suppression of the G₂-M DNA damage checkpointresults in premature mitosis of the cell, and consequently apoptosis. Insome embodiments, provided herein is a method of suppressing the G₂-MDNA damage checkpoint in a cell comprising administering an effectiveamount of a compound of Formula (I), (II) or (III) or a pharmaceuticallyacceptable salt thereof, to the cell. In some embodiments, the G₂-M DNAdamage checkpoint is suppressed in about 40% or more, about 50% or more,about 60% or more, about 70% or more, about 80% or more, about 85% ormore, about 90% or more, about 95% or more, about 96% or more, about 97%or more, about 98% or more, or about 99% or more of cells in a cellpopulation. In some embodiments, the G₂-M DNA damage checkpoint issuppressed in up to about 99%, up to about 98%, up to about 97%, up toabout 96%, up to about 95%, up to about 90%, up to about 85%, or up toabout 80% of cells in the cell population.

In some embodiments, provided herein is a method of inducing prematuremitosis in a cell comprising administering an effective amount of acompound of Formula (I), (II) or (III) or a pharmaceutically acceptablesalt thereof, to the cell. In some embodiments, premature mitosis isinduced in about 40% or more, about 50% or more, about 60% or more,about 70% or more, about 80% or more, about 85% or more, about 90% ormore, about 95% or more, about 96% or more, about 97% or more, about 98%or more, or about 99% or more of cells in a cell population. In someembodiments, premature mitosis is induced in up to about 99%, up toabout 98%, up to about 97%, up to about 96%, up to about 95%, up toabout 90%, up to about 85%, or up to about 80% of cells in the cellpopulation.

In some embodiments, provided herein is a method of inducing apoptosisin a cell comprising administering an effective amount of a compound ofFormula (I), (II) or (III) or a pharmaceutically acceptable saltthereof, to the cell. In some embodiments, apoptosis is induced in about40% or more, about 50% or more, about 60% or more, about 70% or more,about 80% or more, about 85% or more, about 90% or more, about 95% ormore, about 96% or more, about 97% or more, about 98% or more, or about99% or more of cells in a cell population. In some embodiments,apoptosis is induced in up to about 99%, up to about 98%, up to about97%, up to about 96%, up to about 95%, up to about 90%, up to about 85%,or up to about 80% of cells in the cell population.

In some embodiments, provided herein is a method of inhibiting Wee1 in acell comprising administering an effective amount of a compound ofFormula (I), (II) or (III) or a pharmaceutically acceptable saltthereof, to the cell. In some embodiments, Wee1 is inhibited by about10% or more, about 20% or more, about 30% or more, about 40% or more,about 50% or more, about 60% or more, about 70% or more, about 75% ormore, about 80% or more, about 90% or more, about 95% or more, about 96%or more, about 97% or more, about 98% or more, or about 99% or more. Insome embodiments, Wee1 is inhibited up to about 99%, up to about 98%, upto about 97%, up to about 96%, up to about 95%, up to about 90%, up toabout 85%, up to about 80%, up to about 70%, or up to about 60%. In someembodiments, the activity of Wee1 is measured according to a kinaseassay.

In some embodiments, provided herein is a method of inhibiting Wee1comprising contacting Wee1 with an effective amount of a compound ofFormula (I), (II) or (III) or a pharmaceutically acceptable saltthereof. In some embodiments, a compound of Formula (I), (II) or (III)or a pharmaceutically acceptable salt thereof binds to Wee1 with an IC₅₀of less than 1 μM, less than 900 nM, less than 800 nM, less than 700 nM,less than 600 nM, less than 500 nM, less than 400 nM, less than 300 nM,less than 200 nM, less than 100 nM, less than 50 nM, less than 10 nM,less than 5 nM, less than 1 nM, or less than 0.5 nM. In someembodiments, a compound of Formula (I), (II) or (III) or apharmaceutically acceptable salt thereof binds to Wee1 with an IC₅₀between 0.1 nM and 1 nM, between 1 nM and 5 nM, between 5 nM and 10 nM,between 10 nM and 50 nM, between 50 nM and 100 nM, between 100 nM and200 nM, between 200 nM and 300 nM, between 300 nM and 400 nM, between400 nM and 500 nM, between 500 nM and 600 nM, between 600 nM and 700 nM,between 700 nM and 800 nM, between 800 nM and 900 nM, or between 900 nMand 1 μM. In some embodiments, the IC₅₀ is measured according to akinase assay. In some embodiments, the IC₅₀ is measured according to acell cytotoxicity assay.

In some embodiments, provided herein is a method of inhibiting theproliferation of a cell, comprising contacting the cell with aneffective amount of a compound of Formula (I), (II) or (III) or apharmaceutically acceptable salt thereof. In some embodiments, acompound of Formula (I), (II) or (III) or a pharmaceutically acceptablesalt thereof is effective in inhibiting the proliferation of the cellwith an IC₅₀ of less than 5 μM, less than 2 μM, less than 1 μM, lessthan 900 nM, less than 800 nM, less than 700 nM, less than 600 nM, lessthan 500 nM, less than 400 nM, less than 300 nM, less than 200 nM, lessthan 100 nM, or less than 50 nM. In some embodiments, a compound ofFormula (I), (II) or (III) or a pharmaceutically acceptable salt iseffective in inhibiting the proliferation of the cell with an IC₅₀between 10 nM and 20 nM, between 20 nM and 50 nM, between 50 nM and 100nM, between 100 nM and 500 nM, between 500 nM and 1 μM, between 1 μM and2 μM, or between 2 μM and 5 μM. In some embodiments, the IC₅₀ ismeasured according to a cell proliferation assay.

Combination Therapy

As provided herein, the presently disclosed compounds or a salt thereofmay activate the immune system, for example by inducing apoptosis orsuppressing mitosis of cancer cells. Accordingly, the present compoundsor a salt thereof may be used in combination with other anti-canceragents to enhance tumor immunotherapy. In some embodiments, providedherein is a method of treating a disease in an individual comprisingadministering an effective amount of a compound of Formula (I), (II) or(III) or any embodiment, variation or aspect thereof (collectively, acompound of Formula (I), (II) or (III) or the present compounds or thecompounds detailed or described herein) or a pharmaceutically acceptablesalt thereof, and an additional therapeutic agent to the individual. Insome embodiments, the disease is a proliferative disease such as cancer.

In some embodiments, the additional therapeutic agent is a cancerimmunotherapy agent. In some embodiments, the additional therapeuticagent is a chemotherapeutic agent. In some embodiments, the additionaltherapeutic agent is an immunostimulatory agent. In some embodiments,the additional therapeutic agent targets a checkpoint protein (forexample an immune checkpoint inhibitor). In some embodiments, theadditional therapeutic agent is effective to stimulate, enhance orimprove an immune response against a tumor. In some embodiments, theadditional chemotherapeutic agent is a DNA alkylating agent, aplatinum-based chemotherapeutic agent, a kinase inhibitor or a DNAdamage repair (DDR) pathway inhibitor. In some embodiments, theadditional chemotherapeutic agent is a DNA alkylating agent. In someembodiments, the additional chemotherapeutic agent is a platinum-basedchemotherapeutic agent. In some embodiments, the additionalchemotherapeutic agent is a kinase inhibitor. In some embodiments, theadditional chemotherapeutic agent is a DNA damage repair (DDR) pathwayinhibitor.

In another aspect, provided herein is a combination therapy for thetreatment of a disease, such as cancer. In some embodiments, providedherein is a method of treating a disease in an individual comprisingadministering an effective amount of a compound of Formula (I), (II) or(III) or any embodiment, variation or aspect thereof (collectively, acompound of Formula (I), (II) or (III) or the present compounds or thecompounds detailed or described herein) or a pharmaceutically acceptablesalt thereof, in combination with a radiation therapy.

In some embodiments, provided herein is a method of treating a diseasein an individual comprising (a) administering an effective amount of acompound of Formula (I), (II) or (III) or any embodiment, variation oraspect thereof (collectively, Formula (I), (II) or (III)) or apharmaceutically acceptable salt thereof, and (b) administering aneffective amount of an additional chemotherapeutic agent. In someembodiments, the chemotherapeutic agent is a kinase inhibitor or anagent that inhibits one or more DNA damage repair (DDR) pathways. Insome embodiments, a compound of Formula (I), (II) or (III) or apharmaceutically acceptable salt thereof is administered prior to,after, or simultaneously co-administered with the additionalchemotherapeutic agent. In some embodiments, a compound of Formula (I),(II) or (III) or a pharmaceutically acceptable salt thereof isadministered 1 or more hours (such as 2 or more hours, 4 or more hours,8 or more hours, 12 or more hours, 24 or more hours, or 48 or morehours) prior to or after the additional chemotherapeutic agent.

Examples of chemotherapeutic agents that can be used in combination witha compound of Formula (I), (II) or (III) or a pharmaceuticallyacceptable salt thereof include DNA-targeted agents, a DNA alkylatingagent (such as cyclophosphamide, mechlorethamine, chlorambucil,melphalan, dacarbazine, temozolomide or nitrosoureas), a topoisomeraseinhibitor (such as a Topoisomerase I inhibitor (e.g., irinotecan ortopotecan) or a Topoisomerase II inhibitor (e.g., etoposide orteniposide)), an anthracycline (such as daunorubicin, doxorubicin,epirubicin, idarubicin, mitoxantrone, or valrubicin), a histonedeacetylase inhibitor (such as vorinostat or romidepsin), a bromodomaininhibitor, other epigenetic inhibitors, a taxane (such as paclitaxel ordocetaxel), a kinase inhibitor (such as bortezomib, erlotinib,gefitinib, imatinib, vemurafenib, or vismodegib), an anti-angiogenicinhibitor, a nucleotide analog or precursor analog (such as azacitidine,azathioprine, capecitabine, cytarabine, doxifluridine, 5-fluorouracil,gemcitabine, hydroxyurea, mercaptopurine, methotrexate, or tioguanine),or a platinum-based chemotherapeutic agent (such as cisplatin,carboplatin, or oxaliplatin), pemetrexed, or a combination thereof. Insome embodiments, provided herein is a method of treating a disease inan individual comprising (a) administering an effective amount of acompound of Formula (I), (II) or (III) or any embodiment, variation oraspect thereof (collectively, Formula (I), (II) or (III)) or apharmaceutically acceptable salt thereof, and (b) administering aneffective amount of a kinase inhibitor (such as bortezomib, erlotinib,gefitinib, imatinib, vemurafenib, or vismodegib). In some embodiments, acompound of Formula (I), (II) or (III) or a pharmaceutically acceptablesalt thereof is administered prior to, after, or simultaneouslyco-administered with the kinase inhibitor. In some embodiments, acompound of Formula (I), (II) or (III) or a pharmaceutically acceptablesalt thereof is administered 1 or more hours (such as 2 or more hours, 4or more hours, 8 or more hours, 12 or more hours, 24 or more hours, or48 or more hours) prior to or after the kinase inhibitor.

In some embodiments, provided herein is a method of treating a diseasein an individual comprising (a) administering an effective amount of acompound of Formula (I), (II) or (III) or any embodiment, variation oraspect thereof (collectively, Formula (I), (II) or (III)) or apharmaceutically acceptable salt thereof, and (b) administering aneffective amount of a DNA damaging agent. In some embodiments, acompound of Formula (I), (II) or (III) or a pharmaceutically acceptablesalt thereof is administered prior to, after, or simultaneouslyco-administered with the DNA damaging agent. In some embodiments, acompound of Formula (I), (II) or (III) or a pharmaceutically acceptablesalt thereof is administered 1 or more hours (such as 2 or more hours, 4or more hours, 8 or more hours, 12 or more hours, 24 or more hours, or48 or more hours) prior to or after the DNA damaging agent.

In some embodiments, provided herein is a method of treating a diseasein an individual comprising (a) administering an effective amount of acompound of Formula (I), (II) or (III) or any embodiment, variation oraspect thereof (collectively, Formula (I), (II) or (III)) or apharmaceutically acceptable salt thereof, and (b) administering aneffective amount of a DNA alkylating agent (such as cyclophosphamide,mechlorethamine, chlorambucil, melphalan, dacarbazine, temozolomide ornitrosoureas). In some embodiments, a compound of Formula (I), (II) or(III) or a pharmaceutically acceptable salt thereof is administeredprior to, after, or simultaneously co-administered with the DNAalkylating agent. In some embodiments, a compound of Formula (I), (II)or (III) or a pharmaceutically acceptable salt thereof is administered 1or more hours (such as 2 or more hours, 4 or more hours, 8 or morehours, 12 or more hours, 24 or more hours, or 48 or more hours) prior toor after the DNA alkylating agent.

In some embodiments, provided herein is a method of treating a diseasein an individual comprising (a) administering an effective amount of acompound of Formula (I), (II) or (III) or any embodiment, variation oraspect thereof (collectively, Formula (I), (II) or (III)) or apharmaceutically acceptable salt thereof, and (b) administering aneffective amount of a topoisomerase inhibitor (such as a Topoisomerase Iinhibitor (e.g., irinotecan or topotecan) or a Topoisomerase IIinhibitor (e.g., etoposide or teniposide)). In some embodiments, acompound of Formula (I), (II) or (III) or a pharmaceutically acceptablesalt thereof is administered prior to, after, or simultaneouslyco-administered with the topoisomerase inhibitor. In some embodiments, acompound of Formula (I), (II) or (III) or a pharmaceutically acceptablesalt thereof is administered 1 or more hours (such as 2 or more hours, 4or more hours, 8 or more hours, 12 or more hours, 24 or more hours, or48 or more hours) prior to or after the topoisomerase inhibitor.

In some embodiments, provided herein is a method of treating a diseasein an individual comprising (a) administering an effective amount of acompound of Formula (I), (II) or (III) or any embodiment, variation oraspect thereof (collectively, Formula (I), (II) or (III)) or apharmaceutically acceptable salt thereof, and (b) administering aneffective amount of an anthracycline (such as daunorubicin, doxorubicin,epirubicin, idarubicin, mitoxantrone, or valrubicin). In someembodiments, a compound of Formula (I), (II) or (III) or apharmaceutically acceptable salt thereof is administered prior to,after, or simultaneously co-administered with the anthracycline. In someembodiments, a compound of Formula (I), (II) or (III) or apharmaceutically acceptable salt thereof is administered 1 or more hours(such as 2 or more hours, 4 or more hours, 8 or more hours, 12 or morehours, 24 or more hours, or 48 or more hours) prior to or after theanthracycline.

In some embodiments, provided herein is a method of treating a diseasein an individual comprising (a) administering an effective amount of acompound of Formula (I), (II) or (III) or any embodiment, variation oraspect thereof (collectively, Formula (I), (II) or (III)) or apharmaceutically acceptable salt thereof, and (b) administering aneffective amount of a histone deacetylase inhibitor (such as vorinostator romidepsin). In some embodiments, a compound of Formula (I), (II) or(III) or a pharmaceutically acceptable salt thereof is administeredprior to, after, or simultaneously co-administered with the histonedeacetylase inhibitor. In some embodiments, a compound of Formula (I),(II) or (III) or a pharmaceutically acceptable salt thereof isadministered 1 or more hours (such as 2 or more hours, 4 or more hours,8 or more hours, 12 or more hours, 24 or more hours, or 48 or morehours) prior to or after the histone deacetylase inhibitor.

In some embodiments, provided herein is a method of treating a diseasein an individual comprising (a) administering an effective amount of acompound of Formula (I), (II) or (III) or any embodiment, variation oraspect thereof (collectively, Formula (I), (II) or (III)) or apharmaceutically acceptable salt thereof, and (b) administering aneffective amount of a taxane (such as paclitaxel or docetaxel). In someembodiments, a compound of Formula (I), (II) or (III) or apharmaceutically acceptable salt thereof is administered prior to,after, or simultaneously co-administered with the taxane. In someembodiments, a compound of Formula (I), (II) or (III) or apharmaceutically acceptable salt thereof is administered 1 or more hours(such as 2 or more hours, 4 or more hours, 8 or more hours, 12 or morehours, 24 or more hours, or 48 or more hours) prior to or after thetaxane.

In some embodiments, provided herein is a method of treating a diseasein an individual comprising (a) administering an effective amount of acompound of Formula (I), (II) or (III) or any embodiment, variation oraspect thereof (collectively, Formula (I), (II) or (III)) or apharmaceutically acceptable salt thereof, and (b) administering aneffective amount of a nucleotide analog or precursor analog (such asazacitidine, azathioprine, capecitabine, cytarabine, doxifluridine,5-fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate,or tioguanine). In some embodiments, a compound of Formula (I), (II) or(III) or a pharmaceutically acceptable salt thereof is administeredprior to, after, or simultaneously co-administered with the nucleotideanalog or precursor analog. In some embodiments, a compound of Formula(I), (II) or (III) or a pharmaceutically acceptable salt thereof isadministered 1 or more hours (such as 2 or more hours, 4 or more hours,8 or more hours, 12 or more hours, 24 or more hours, or 48 or morehours) prior to or after the nucleotide analog or precursor analog.

In some embodiments, provided herein is a method of treating a diseasein an individual comprising (a) administering an effective amount of acompound of Formula (I), (II) or (III) or any embodiment, variation oraspect thereof (collectively, Formula (I), (II) or (III)) or apharmaceutically acceptable salt thereof, and (b) administering aneffective amount of a platinum-based chemotherapeutic agent (such ascisplatin, carboplatin, or oxaliplatin). In some embodiments, a compoundof Formula (I), (II) or (III) or a pharmaceutically acceptable saltthereof is administered prior to, after, or simultaneouslyco-administered with the platinum-based chemotherapeutic agent. In someembodiments, a compound of Formula (I), (II) or (III) or apharmaceutically acceptable salt thereof is administered 1 or more hours(such as 2 or more hours, 4 or more hours, 8 or more hours, 12 or morehours, 24 or more hours, or 48 or more hours) prior to or after theplatinum-based chemotherapeutic agent.

In some embodiments, provided herein is a method of treating a diseasein an individual comprising (a) administering an effective amount of acompound of Formula (I), (II) or (III) or any embodiment, variation oraspect thereof (collectively, Formula (I), (II) or (III)) or apharmaceutically acceptable salt thereof, and (b) administering aneffective amount of pemetrexed. In some embodiments, a compound ofFormula (I), (II) or (III) or a pharmaceutically acceptable salt thereofis administered prior to, after, or simultaneously co-administered withthe pemetrexed. In some embodiments, a compound of Formula (I), (II) or(III) or a pharmaceutically acceptable salt thereof is administered 1 ormore hours (such as 2 or more hours, 4 or more hours, 8 or more hours,12 or more hours, 24 or more hours, or 48 or more hours) prior to orafter the pemetrexed.

In some embodiments, provided herein is a method of treating a diseasein an individual comprising (a) administering an effective amount of acompound of Formula (I), (II) or (III) or any embodiment, variation oraspect thereof (collectively, Formula (I), (II) or (III)) or apharmaceutically acceptable salt thereof, and (b) administering aneffective amount of a DDR pathway inhibitor. In some embodiments, acompound of Formula (I), (II) or (III) or a pharmaceutically acceptablesalt thereof is administered prior to, after, or simultaneouslyco-administered with the DDR pathway inhibitor. In some embodiments, acompound of Formula (I), (II) or (III) or a pharmaceutically acceptablesalt thereof is administered 1 or more hours (such as 2 or more hours, 4or more hours, 8 or more hours, 12 or more hours, 24 or more hours, or48 or more hours) prior to or after the DDR pathway inhibitor. Examplesof inhibitors of the DDR pathway include poly(ADP-ribose) polymerase(PARP) inhibitors (such as olaparib, rucaparib, niraparib, ortalazoparib), ataxia telangiectasia mutated (ATM) protein inhibitors,ataxia telangiectasia and Rad3-related (ATR) protein inhibitors,checkpoint kinase 1 (Chk1) inhibitors, or combinations thereof.

In some embodiments, provided herein is a method of treating a diseasein an individual comprising (a) administering an effective amount of acompound of Formula (I), (II) or (III) or any embodiment, variation oraspect thereof (collectively, Formula (I), (II) or (III)) or apharmaceutically acceptable salt thereof, and (b) administering aneffective amount of a PARP inhibitor (such as olaparib, rucaparib,niraparib, or talazoparib). In some embodiments, a compound of Formula(I), (II) or (III) or a pharmaceutically acceptable salt thereof isadministered prior to, after, or simultaneously co-administered with thePARP inhibitor. In some embodiments, a compound of Formula (I), (II) or(III) or a pharmaceutically acceptable salt thereof is administered 1 ormore hours (such as 2 or more hours, 4 or more hours, 8 or more hours,12 or more hours, 24 or more hours, or 48 or more hours) prior to orafter the PARP inhibitor.

In some embodiments, provided herein is a method of treating a diseasein an individual comprising (a) administering an effective amount of acompound of Formula (I), (II) or (III) or any embodiment, variation oraspect thereof (collectively, Formula (I), (II) or (III)) or apharmaceutically acceptable salt thereof, and (b) administering aneffective amount of an ATM protein inhibitor. In some embodiments, acompound of Formula (I), (II) or (III) or a pharmaceutically acceptablesalt thereof is administered prior to, after, or simultaneouslyco-administered with the ATM protein inhibitor. In some embodiments, acompound of Formula (I), (II) or (III) or a pharmaceutically acceptablesalt thereof is administered 1 or more hours (such as 2 or more hours, 4or more hours, 8 or more hours, 12 or more hours, 24 or more hours, or48 or more hours) prior to or after the ATM protein inhibitor.

In some embodiments, provided herein is a method of treating a diseasein an individual comprising (a) administering an effective amount of acompound of Formula (I), (II) or (III) or any embodiment, variation oraspect thereof (collectively, Formula (I), (II) or (III)) or apharmaceutically acceptable salt thereof, and (b) administering aneffective amount of an ATR protein inhibitor. In some embodiments, acompound of Formula (I), (II) or (III) or a pharmaceutically acceptablesalt thereof is administered prior to, after, or simultaneouslyco-administered with the ATR protein inhibitor. In some embodiments, acompound of Formula (I), (II) or (III) or a pharmaceutically acceptablesalt thereof is administered 1 or more hours (such as 2 or more hours, 4or more hours, 8 or more hours, 12 or more hours, 24 or more hours, or48 or more hours) prior to or after the ATR protein inhibitor.

In some embodiments, provided herein is a method of treating a diseasein an individual comprising (a) administering an effective amount of acompound of Formula (I), (II) or (III) or any embodiment, variation oraspect thereof (collectively, Formula (I), (II) or (III)) or apharmaceutically acceptable salt thereof, and (b) administering aneffective amount of an Chk1 inhibitor. In some embodiments, a compoundof Formula (I), (II) or (III) or a pharmaceutically acceptable saltthereof is administered prior to, after, or simultaneouslyco-administered with the Chk1 inhibitor. In some embodiments, a compoundof Formula (I), (II) or (III) or a pharmaceutically acceptable saltthereof is administered 1 or more hours (such as 2 or more hours, 4 ormore hours, 8 or more hours, 12 or more hours, 24 or more hours, or 48or more hours) prior to or after the Chk1 inhibitor.

In another aspect, provided herein is a combination therapy in which acompound of Formula (I), (II) or (III) or a salt thereof iscoadministered (which may be separately or simultaneously) with one ormore additional agents that are effective in stimulating immuneresponses to thereby further enhance, stimulate or upregulate immuneresponses in a subject. For example, provided is a method forstimulating an immune response in a subject comprising administering tothe subject a compound of Formula (I), (II) or (III) or a salt thereofand one or more immunostimulatory antibodies, such as an anti-PD-1antibody, an anti-PD-L1 antibody and/or an anti-CTLA-4 antibody, suchthat an immune response is stimulated in the subject, for example toinhibit tumor growth. In one embodiment, the subject is administered acompound of Formula (I), (II) or (III) or a salt thereof and ananti-PD-1 antibody. In another embodiment, the subject is administered acompound of Formula (I), (II) or (III) or a salt thereof and ananti-PD-L1 antibody. In yet another embodiment, the subject isadministered a compound of formula (I) or a salt thereof and ananti-CTLA-4 antibody. In another embodiment, the immunostimulatoryantibody (e.g., anti-PD-1, anti-PD-L1 and/or anti-CTLA-4 antibody) is ahuman antibody. Alternatively, the immunostimulatory antibody can be,for example, a chimeric or humanized antibody (e.g., prepared from amouse anti-PD-1, anti-PD-L1 and/or anti-CTLA-4 antibody).

In one embodiment, the present disclosure provides a method for treatinga proliferative disease (e.g., cancer), comprising administering acompound of Formula (I), (II) or (III) or a salt thereof and ananti-PD-1 antibody to a subject. In further embodiments, a compound ofFormula (I), (II) or (III) or a salt thereof is administered at asubtherapeutic dose, the anti-PD-1 antibody is administered at asubtherapeutic dose, or both are administered at a subtherapeutic dose.In another embodiment, the present disclosure provides a method foraltering an adverse event associated with treatment of ahyperproliferative disease with an immunostimulatory agent, comprisingadministering a compound of Formula (I), (II) or (III) or a salt thereofand a subtherapeutic dose of anti-PD-1 antibody to a subject. In certainembodiments, the subject is human. In certain embodiments, the anti-PD-1antibody is a human sequence monoclonal antibody.

In one embodiment, the present invention provides a method for treatinga hyperproliferative disease (e.g., cancer), comprising administering acompound of Formula (I), (II) or (III) or a salt thereof and ananti-PD-L1 antibody to a subject. In further embodiments, a compound ofFormula (I), (II) or (III) or a salt thereof is administered at asubtherapeutic dose, the anti-PD-L1 antibody is administered at asubtherapeutic dose, or both are administered at a subtherapeutic dose.In another embodiment, the present invention provides a method foraltering an adverse event associated with treatment of ahyperproliferative disease with an immunostimulatory agent, comprisingadministering a compound of Formula (I), (II) or (III) or a salt thereofand a subtherapeutic dose of anti-PD-L1 antibody to a subject. Incertain embodiments, the subject is human. In certain embodiments, theanti-PD-L1 antibody is a human sequence monoclonal antibody.

In certain embodiments, the combination of therapeutic agents discussedherein can be administered concurrently as a single composition in apharmaceutically acceptable carrier, or concurrently as separatecompositions each in a pharmaceutically acceptable carrier. In anotherembodiment, the combination of therapeutic agents can be administeredsequentially. For example, an anti-CTLA-4 antibody and a compound ofFormula (I), (II) or (III) or a salt thereof can be administeredsequentially, such as anti-CTLA-4 antibody being administered first anda compound of Formula (I), (II) or (III) or a salt thereof second, or acompound of formula Formula (I), (II) or (III) or a salt thereof beingadministered first and anti-CTLA-4 antibody second. Additionally oralternatively, an anti-PD-1 antibody and a compound of Formula (I), (II)or (III) or a salt thereof can be administered sequentially, such asanti-PD-1 antibody being administered first and a compound of Formula(I), (II) or (III) or a salt thereof second, or a compound of Formula(I), (II) or (III) or a salt thereof being administered first andanti-PD-1 antibody second. Additionally or alternatively, an anti-PD-L1antibody and a compound of Formula (I), (II) or (III) or a salt thereofcan be administered sequentially, such as anti-PD-L1 antibody beingadministered first and a compound of Formula (I), (II) or (III) or asalt thereof second, or a compound of Formula (I), (II) or (III) or asalt thereof being administered first and anti-PD-L1 antibody second.

Furthermore, if more than one dose of the combination therapy isadministered sequentially, the order of the sequential administrationcan be reversed or kept in the same order at each time point ofadministration, sequential administrations can be combined withconcurrent administrations, or any combination thereof.

Optionally, the combination of a compound of Formula (I), (II) or (III)or a salt thereof can be further combined with an immunogenic agent,such as cancerous cells, purified tumor antigens (including recombinantproteins, peptides, and carbohydrate molecules), cells, and cellstransfected with genes encoding immune stimulating cytokines.

A compound of Formula (I), (II) or (III) or a salt thereof can also befurther combined with standard cancer treatments. For example, acompound of Formula (I), (II) or (III) or a salt thereof can beeffectively combined with chemotherapeutic regimes. In these instances,it is possible to reduce the dose of other chemotherapeutic reagentadministered with the combination of the instant disclosure (Mokyr etal. (1998) Cancer Research 58: 5301-5304). Other combination therapieswith a compound of Formula (I), (II) or (III) or a salt thereof includeradiation, surgery, or hormone deprivation. Angiogenesis inhibitors canalso be combined with a compound of Formula (I), (II) or (III) or a saltthereof. Inhibition of angiogenesis leads to tumor cell death, which canbe a source of tumor antigen fed into host antigen presentationpathways.

In another example, a compound of Formula (I), (II) or (III) or a saltthereof can be used in conjunction with anti-neoplastic antibodies. Byway of example and not wishing to be bound by theory, treatment with ananti-cancer antibody or an anti-cancer antibody conjugated to a toxincan lead to cancer cell death (e.g., tumor cells) which would potentiatean immune response mediated by CTLA-4, PD-1, PD-L1 or a compound ofFormula (I), (II) or (III) or a salt thereof. In an exemplaryembodiment, a treatment of a hyperproliferative disease (e.g., a cancertumor) can include an anti-cancer antibody in combination with acompound of Formula (I), (II) or (III) or a salt thereof and anti-CTLA-4and/or anti-PD-1 and/or anti-PD-L1 antibodies, concurrently orsequentially or any combination thereof, which can potentiate anti-tumorimmune responses by the host. Other antibodies that can be used toactivate host immune responsiveness can be further used in combinationwith a compound of Formula (I), (II) or (III) or a salt thereof.

In some embodiments, a compound of Formula (I), (II) or (III) or a saltthereof can be combined with an anti-CD73 therapy, such as an anti-CD73antibody.

In yet further embodiments, a compound of Formula (I), (II) or (III) ora salt thereof is administered in combination with another Wee1inhibitor.

Dosing and Method of Administration

The dose of a compound administered to an individual (such as a human)may vary with the particular compound or salt thereof, the method ofadministration, and the particular disease, such as type and stage ofcancer, being treated. In some embodiments, the amount of the compoundor salt thereof is a therapeutically effective amount.

The effective amount of the compound may in one aspect be a dose ofbetween about 0.01 and about 100 mg/kg. Effective amounts or doses ofthe compounds of the invention may be ascertained by routine methods,such as modeling, dose escalation, or clinical trials, taking intoaccount routine factors, e.g., the mode or route of administration ordrug delivery, the pharmacokinetics of the agent, the severity andcourse of the disease to be treated, the subject's health status,condition, and weight. An exemplary dose is in the range of about fromabout 0.7 mg to 7 g daily, or about 7 mg to 350 mg daily, or about 350mg to 1.75 g daily, or about 1.75 to 7 g daily.

Any of the methods provided herein may in one aspect compriseadministering to an individual a pharmaceutical composition thatcontains an effective amount of a compound provided herein or a saltthereof and a pharmaceutically acceptable excipient.

A compound or composition of the invention may be administered to anindividual in accordance with an effective dosing regimen for a desiredperiod of time or duration, such as at least about one month, at leastabout 2 months, at least about 3 months, at least about 6 months, or atleast about 12 months or longer, which in some variations may be for theduration of the individual's life. In one variation, the compound isadministered on a daily or intermittent schedule. The compound can beadministered to an individual continuously (for example, at least oncedaily) over a period of time. The dosing frequency can also be less thanonce daily, e.g., about a once weekly dosing. The dosing frequency canbe more than once daily, e.g., twice or three times daily. The dosingfrequency can also be intermittent, including a ‘drug holiday’ (e.g.,once daily dosing for 7 days followed by no doses for 7 days, repeatedfor any 14 day time period, such as about 2 months, about 4 months,about 6 months or more). Any of the dosing frequencies can employ any ofthe compounds described herein together with any of the dosagesdescribed herein.

The compounds provided herein or a salt thereof may be administered toan individual via various routes, including, e.g., intravenous,intramuscular, subcutaneous, oral and transdermal. A compound providedherein can be administered frequently at low doses, known as ‘metronomictherapy,’ or as part of a maintenance therapy using compound alone or incombination with one or more additional drugs. Metronomic therapy ormaintenance therapy can comprise administration of a compound providedherein in cycles. Metronomic therapy or maintenance therapy can compriseintra-tumoral administration of a compound provided herein.

In one aspect, the invention provides a method of treating cancer in anindividual by parenterally administering to the individual (e.g., ahuman) an effective amount of a compound or salt thereof. In someembodiments, the route of administration is intravenous, intra-arterial,intramuscular, or subcutaneous. In some embodiments, the route ofadministration is oral. In still other embodiments, the route ofadministration is transdermal.

The invention also provides compositions (including pharmaceuticalcompositions) as described herein for the use in treating, preventing,and/or delaying the onset and/or development of cancer and other methodsdescribed herein. In certain embodiments, the composition comprises apharmaceutical formulation which is present in a unit dosage form.

Also provided are articles of manufacture comprising a compound of thedisclosure or a salt thereof, composition, and unit dosages describedherein in suitable packaging for use in the methods described herein.Suitable packaging is known in the art and includes, for example, vials,vessels, ampules, bottles, jars, flexible packaging and the like. Anarticle of manufacture may further be sterilized and/or sealed.

Kits

The present disclosure further provides kits for carrying out themethods of the invention, which comprises one or more compoundsdescribed herein or a composition comprising a compound describedherein. The kits may employ any of the compounds disclosed herein. Inone variation, the kit employs a compound described herein or a saltthereof. The kits may be used for any one or more of the uses describedherein, and, accordingly, may contain instructions for the treatment ofcancer.

Kits generally comprise suitable packaging. The kits may comprise one ormore containers comprising any compound described herein. Each component(if there is more than one component) can be packaged in separatecontainers or some components can be combined in one container wherecross-reactivity and shelf life permit.

The kits may be in unit dosage forms, bulk packages (e.g., multi-dosepackages) or sub-unit doses. For example, kits may be provided thatcontain sufficient dosages of a compound as disclosed herein and/or anadditional pharmaceutically active compound useful for a diseasedetailed herein to provide effective treatment of an individual for anextended period, such as any of a week, 2 weeks, 3 weeks, 4 weeks, 6weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9months, or more. Kits may also include multiple unit doses of thecompounds and instructions for use and be packaged in quantitiessufficient for storage and use in pharmacies (e.g., hospital pharmaciesand compounding pharmacies).

The kits may optionally include a set of instructions, generally writteninstructions, although electronic storage media (e.g., magnetic disketteor optical disk) containing instructions are also acceptable, relatingto the use of component(s) of the methods of the present invention. Theinstructions included with the kit generally include information as tothe components and their administration to an individual.

The invention can be further understood by reference to the followingexamples, which are provided by way of illustration and are not meant tobe limiting.

EXAMPLES Example S-1: Synthesis of1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(Compound No. 1.1)

Step-1: Synthesis of 2-(6-bromo-3-fluoropyridin-2-yl)propan-2-ol: To asolution of 2-bromo-5-fluoropyridine (2 g, 11.36 mmol) in diethyl-ether(30 mL) was slowly added n-butyl lithium (2.5 M in hexane, 7.8 mL, 12.49mmol) at −78° C. under a nitrogen atmosphere. The resulting yellowreaction mixture was stirred at −78° C. for 2 hours and dry acetone (1.0mL, 13.63 mmol) was added over 30 minutes. Stirring was continued at−78° C. for 1 hour. HCl (2N, 50 mL) was added and the reaction mixturewas warmed to 0° C. The pH of the mixture was adjusted to 7 with 2N HClsolution. The reaction mixture was diluted with ethyl acetate and washedwith brine, dried over sodium sulfate to afford crude product, which waspurified by flash chromatography [silica gel 100-200 mesh; elution 0-50%EtOAc in hexane] to afford the desired product2-(6-bromo-3-fluoro-pyridin-2-yl)-propan-2-ol (1.5 g, 56.81%) as yellowsemi solid. LCMS: 234.1 [M+2]⁺.

Step-2: Synthesis of 6-bromo-3-fluoro-2-(2-fluoropropan-2-yl)pyridine:To a stirred solution of 2-(6-bromo-3-fluoropyridin-2-yl)propan-2-ol(0.500 g, 2.14 mmol, 1.0 eq) in DCM (10 ml), DAST (0.38 mL, 2.36 mmol,1.1 eq) was added at −78° C. The reaction mixture was stirred at RT for12 h. After completion of reaction, the reaction mixture was quenchedwith saturated sodium bicarbonate solution and was extracted with EtOAc(50 mL×2). The combined organic layer was washed with water (50 me) andbrine solution (50 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure to afford crude product, which waspurified by flash chromatography [silica gel 100-200 mesh; elution 0-10%EtOAc in hexane] to afford the desired compound, (200 mg, 39.66%) ascolorless liquid. LCMS: 236.1 [+M+2]⁺.

Step-3: Synthesis of1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:To a stirred solution of2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(224 mg, 1.0 mmol, 1.0 eq) and6-bromo-3-fluoro-2-(2-fluoropropan-2-yl)pyridine (236 mg, 1.0 mmol, 1.0eq) in dioxane (10 mL) were added potassium carbonate (276 mg, 2.0 mmol,2.0 eq) and the resulting mixture was purged with nitrogen for 10 minfollowed by addition of copper iodide (38 mg, 0.2 mmol, 0.2 eq), andN,N′-dimethylethylenediamine (DMEDA) (0.05 mL, 0.4 mmol, 0.4 eq) andagain purged with nitrogen for 10 min, then stirred at 900 C overnight.After completion of reaction, the reaction mixture was diluted withwater and extracted with EtOAc (50 mL×2). Combined organic layer waswashed with water (50 mL) and brine solution (50 mL), dried overanhydrous sodium sulphate and concentrated under reduced pressure toafford crude product, which was purified by flash chromatography toafford the desired compound (200 mg, 52.6%) as an off-white solid. LCMS:380.2 [M+1]⁺.

Step-4: Synthesis of tert-butyl6-((1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(190 mg, 0.5 mmol, 1.0 eq) in toluene (2.0 mL) was added m-CPBA (245 mg,1.0 mmol, 2.0 eq) and allowed to stir at RT for 1 h. Tert-butyl6-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (149 mg, 0.6 mmol, 1.2eq) and DIPEA (0.44 mL, 2.5 mmol, 5.0 eq) were added and allowed to stirat RT overnight. After completion of reaction, the reaction mixture wasdiluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layer was washed with water (50 mL) and brine solution (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-50% EtOAc in hexane]to afford the desired compound (100 mg, 34.6%) as an off-white solid.LCMS: 580.3 [M+1]⁺.

Step-5: Synthesis of1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:Tert-butyl6-((1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate(100 mg, 0.17 mmol, 1.0 eq) was dissolved in dioxane (1.0 mL), followedby dropwise addition of 4.0 M-HCl (1.0 mL) and allowed to stir at RT 2h. After completion of reaction, solvent was evaporated to give thecrude product, which was purified reverse phase chromatography to affordthe desired compound (45 mg, 55.5%) as an off-white solid. LCMS: 457.3[M+1]⁺; ¹H NMR (400 MHz, DMSO-d₆, Formate salt): δ 10.24 (br s, 1H) 8.82(s, 1H) 8.69 (br s, 1H) 8.39 (d, J=7.45 Hz, 1H) 8.28 (br s, 1H) 8.10 (t,J=9.65 Hz, 1H) 7.96 (d, J=8.33 Hz, 2H) 7.55 (br s, 1H) 7.35-7.49 (m, 2H)7.03 (d, J=8.33 Hz, 1H) 4.11-4.22 (m, 2H) 3.96 (br s, 2H) 3.08-3.17 (m,2H) 2.79 (br s, 2H) 1.77 (s, 3H) 1.71 (s, 3H) 1.33 (d, J=6.58 Hz, 6H).

Example S-2: Synthesis of2-cyclopropyl-1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(Compound No. 1.2)

Step-1: Synthesis of2-cyclopropyl-1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:To a stirred solution of2-cyclopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(230 mg, 1.03 mmol, 1.0 eq) and6-bromo-3-fluoro-2-(2-fluoropropan-2-yl)pyridine (291 mg, 1.24 mmol, 1.2eq) in dioxane (5 mL) was added potassium carbonate (285 mg, 2.06 mmol,2.0 eq) and the resulting mixture was purged with nitrogen for 10 minfollowed by addition of copper iodide (39.4 mg, 0.20 mmol, 0.2 eq), andN,N′-dimethylethylenediamine (DMEDA) (0.04 mL, 0.41 mmol, 0.4 eq) andagain purged with nitrogen for 10 min, then stirred at 130° C.overnight. After completion of reaction, the reaction mixture wasdiluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layer was washed with water (50 mL) and brine solution (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-50% EtOAc in hexane]to afford the desired product (180 mg, 48.74%) as yellow semi solid.LCMS: 378.11 [M+1]⁺.

Step-2: Synthesis of tert-butyl6-((2-cyclopropyl-1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of2-cyclopropyl-1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(190 mg, 0.50 mmol, 1.0 eq) in toluene (3.0 mL) was added m-CPBA (174mg, 1.00 mmol, 2.0 eq) and allowed to stir at RT for 60 minutes.Tert-butyl 6-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (150 mg,0.60 mmol, 1.2 eq) and DIPEA (0.4 mL, 0.55 mmol, 4.0 eq) were added andallowed to stir at RT overnight. After completion of reaction, thereaction mixture was diluted with water and extracted with EtOAc (50mL×2). The combined organic layer was washed with water (50 mL) andbrine solution (50 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure to afford crude product, which waspurified by flash chromatography [silica gel 100-200 mesh; elution 0-50%EtOAc in hexane] to afford the desired compound (130 mg, 44.77%) aslight yellow solid. LCMS: 578.3 [M+1]⁺.

Step-3: Synthesis of2-cyclopropyl-1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-onedihydrochloride: Tert-butyl6-((2-cyclopropyl-1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate(130 mg, 0.22 mmol, 1.0 eq) was dissolved in dioxane (2 mL), followed bydropwise addition of 4.0 M-HCl in dioxane (3 mL) and allowed to stir atRT for 2 h. After completion of reaction, the reaction mixture wasfiltered and dried under reduced pressure; crude product obtained waspurified by reverse phase chromatography to afford the desired compound(2 mg, 20.48%) as an off-white solid. LCMS: 478.3 [M+1]⁺; ¹H NMR (400MHz, DMSO-d₆, HCl salt): δ 10.17 (br s, 1H) 8.79 (s, 1H) 8.31 (br s, 1H)8.04-8.18 (m, 2H) 7.96 (dd, 1H) 7.56 (br s, 1H) 7.40 (d, J=7.89 Hz, 1H)7.02 (d, 1H) 3.91 (br s, 2H) 3.14 (br s, 2H) 3.07 (br s, 2H) 2.75 (br s,2H) 1.77 (s, 3H) 1.72 (s, 3H).

Example S-3: Synthesis of1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(Compound No. 1.4)

Step-1: Synthesis tert-butyl7-((1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(130 mg, 0.3 mmol, 1.0 eq) in toluene (2.0 mL) was added m-CPBA (148 mg,0.6 mmol, 2.0 eq) and allowed to stir at RT for 1 h. Tert-butyl7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (149 mg, 0.6 mmol, 1.2eq) and DIPEA (0.27 mL, 1.5 mmol, 5.0 eq) were added and allowed to stirat RT overnight. After completion of reaction, the reaction mixture wasdiluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layer was washed with water (50 mL) and brine solution (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-50% EtOAc in hexane]to afford the desired compound (120 mg, 68.9%) as an off-white solid.LCMS: 580.4 [M+1]⁺.

Step-2: Synthesis of1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:Tert-butyl7-((1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate(120 mg, 0.21 mmol, 1.0 eq) was dissolved in dioxane (2.0 mL), followedby dropwise addition of 4.0 M-HCl in dioxane (2.0 mL) and allowed tostir at RT for 1 h. After completion of reaction, solvent was evaporatedto give the crude product, which was purified reverse phasechromatography to afford the desired compound (32 mg, 32.0%) as anoff-white solid as formate salt. LCMS: 480.5 [M+1]⁺; ¹H NMR (400 MHz,DMSO-d₆, Formate salt): δ 10.25 (br sbr s, 1H) 8.82 (s, 1H) 8.28 (br s,1H) 8.05-8.13 (m, 1H) 7.97 (d, J=6.58 Hz, 1H) 7.52 (br sbr s, 1H) 7.38(d, J=8.33 Hz, 1H) 7.06 (d, J=7.89 Hz, 1H) 4.12-4.19 (m, 2H) 3.96 (brsbr s, 2H) 3.07 (br sbr s, 2H) 2.73 (br sbr s, 2H) 1.77 (s, 3H) 1.71 (s,3H) 1.34 (d, J=6.58 Hz, 6H).

Example S-4: Synthesis of1-(2-cyclopropylthiazol-4-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(Compound No. 1.464)

Step-1: Synthesis of 4-bromo-2-cyclopropylthiazole: To a stirredsolution of 2,4-dibromothiazole (1.0 g, 4.11 mmol, 1.0 eq) andcyclopropylboronic acid (424 mg, 4.93 mmol, 1.2 eq) in dioxane (12 mL)were added K₃PO₄ (1.11 g, 8.08 mmol, 2 eq) and the resulting mixture waspurged with nitrogen for 10 min, followed by addition of Xanthphos (119mg, 0.20 mmol, 0.2 eq), and Pd(OAc)₂ (46 mg, 0.20 mmol, 0.4 eq) andagain purged with nitrogen for 10 min, stirred at 90° C. overnight.After completion of reaction, the reaction mixture was diluted withwater and extracted with EtOAc (250 mL×2). The combined organic layerswere washed with water (250 mL) and brine solution (250 mL), dried overanhydrous sodium sulphate and concentrated under reduced pressure toafford crude product, which was purified by flash chromatography toafford the desired compound (600 mg, 71.42%) as an off-white solid.LCMS: 204.1 [M+1]⁺.

Step-2: Synthesis of1-(2-cyclopropylthiazol-4-yl)-2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:To a stirred solution of2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(659 mg, 2.93 mmol, 1.0 eq) and 4-bromo-2-cyclopropylthiazole (600 mg,2.93 mmol, 1.0 eq) in dioxane (12 mL) were added potassium carbonate(810 mg, 5.86 mmol, 2.0 eq) and the resulting mixture was purged withnitrogen for 10 min, followed by addition of copper iodide (112 mg, 0.58mmol, 0.2 eq), and N,N′-dimethylethylenediamine (DMEDA) (0.13 mL, 1.17mmol, 0.4 eq) and again purged with nitrogen for 10 min, then stirred at900 Covernight. After completion of reaction, the reaction mixture wasdiluted with water and extracted with EtOAc (250 mL×2). The combinedorganic layers were washed with water (250 mL) and brine solution (250mL), dried over anhydrous sodium sulphate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography to afford the desired compound (256 mg, 25.07%) as anoff-white solid. LCMS: 348.1 [M+1]⁺.

Step-3: Synthesis of tert-butyl6-((1-(2-cyclopropylthiazol-4-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirring solution of1-(2-cyclopropylthiazol-4-yl)-2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(128 mg, 0.36 mmol, 1 eq) in toluene (3.0 mL) was added m-CPBA (127 mg,0.73 mmol, 2.0 eq) and allowed to stir at RT for 1 h. Tert-butyl6-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (91 mg, 0.36 mmol, 1.0eq) and DIPEA (0.25 mL, 1.47 mmol, 4.0 eq) were added and allowed tostir at RT overnight. After completion of reaction, the reaction mixturewas diluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layer was washed with water (50 mL) and brine solution (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-50% EtOAc in hexane]to afford the desired compound (86 mg, 42.62%) as an off-white solid.LCMS: 548.3 [M+1]⁺.

Step-4: Synthesis of1-(2-cyclopropylthiazol-4-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-onehydrochloride: Tert-butyl6-((1-(2-cyclopropylthiazol-4-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate(86 mg, 0.15 mmol, 1.0 eq) was dissolved in dioxane (1 mL), followed bydropwise addition of 4.0 M-HCl in dioxane (1 mL) and allowed to stir atRT for 1 h. After completion of reaction, the reaction mixture wasfiltered and dried under reduced pressure to afford the desired compound(30 mg, 39.47%) as an off-white solid. LCMS: 448.3 [M+1]⁺; ¹H NMR (400MHz, DMSO-d₆, HCl salt): δ 10.27 (br sbr s, 1H) 9.20 (br sbr s, 2H) 8.84(s, 1H) 7.90 (s, 1H) 7.67 (br sbr s, 1H) 7.48 (d, J=7.89 Hz, 1H) 7.11(d, J=8.77 Hz, 1H) 4.28 (dt,J=13.59, 6.80 Hz, 1H) 4.19 (br sbr s, 2H)3.35 (br sbr s, 2H) 2.92 (br sbr s, 2H) 1.17-1.27 (m, 6H) 1.07-1.17 (m,2H) 0.88-0.97 (m, 2H).

Example S-5: Synthesis of1-(2-cyclopropylthiazol-4-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(Compound No. 1.465)

Step-1: Synthesis of tert-butyl7-((1-(2-cyclopropylthiazol-4-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirring solution of1-(2-cyclopropylthiazol-4-yl)-2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(128 mg, 0.36 mmol, 1 eq) in toluene (3.0 mL) was added m-CPBA (127 mg,0.73 mmol, 2.0 eq) and allowed to stir at RT for 1 h. Tert-butyl7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (91 mg, 0.36 mmol, 1.0eq) and DIPEA (0.25 mL, 1.47 mmol, 4.0 eq) were added and allowed tostir at RT overnight. After completion of reaction, the reaction mixturewas diluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layer was washed with water (50 mL) and brine solution (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-50% EtOAc in hexane]to afford the desired compound (86 mg, 51.54%) as an off-white solid.LCMS: 548.3 [M+1]⁺.

Step-2: Synthesis of1-(2-cyclopropylthiazol-4-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-onehydrochloride: Tert-butyl7-((1-(2-cyclopropylthiazol-4-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate(104 mg, 0.18 mmol, 1.0 eq) was dissolved in dioxane (1.5 mL), followedby dropwise addition of 4.0 M-HCl in dioxane (1.5 mL) and allowed tostir at RT for 1 h. After completion of reaction, the reaction mixturewas filtered and dried under reduced pressure to afford the desiredcompound (20 mg, 17.68%) as an off-white solid. LCMS: 448.3 [M+1]⁺; ¹HNMR (400 MHz, DMSO-d₆, HCl salt): δ 10.28 (br s, 1H) 9.40 (br s, 2H)8.83 (s, 1H) 7.90 (s, 1H) 7.65 (br., 1H) 7.47 (d, J=7.89 Hz, 1H) 7.12(d, J=8.77 Hz, 1H) 4.21-4.33 (m, 2H) 4.17 (br s, 2H) 3.33 (br s, 2H)2.94 (br s, 2H) 1.22 (d, J=7.02 Hz, 6H) 1.16 (dd, J=8.11, 2.41 Hz, 2H)0.94 (br s, 2H).

Example S-6: Synthesis of2-cyclopropyl-6-((1,1-dimethyl-1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one (Compound No. 1.5)

Step-1: Synthesis of 2-cyclopropyl-1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:To a stirred solution of(2-cyclopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(230 mg, 1.03 mmol, 1.0 eq) and6-bromo-3-fluoro-2-(2-fluoropropan-2-yl)pyridine (291 mg, 1.24 mmol, 1.2eq) in dioxane (5 mL) was added potassium carbonate (285 mg, 2.06 mmol,2.0 eq) and the resulting mixture was purged with nitrogen for 10 minfollowed by addition of copper iodide (40 mg, 0.26 mmol, 0.2 eq), andN,N′-dimethylethylenediamine (DMEDA) (0.04 mL, 0.413 mmol, 0.4 eq) andagain purged with nitrogen for 10 min, stirred at 130° C. overnight.After completion of reaction, the reaction mixture was diluted withwater and extracted with EtOAc (50 mL×2). The combined organic layer waswashed with water (50 mL) and brine solution (50 mL), dried overanhydrous sodium sulfate and concentrated under reduced pressure toafford crude product, which was purified by flash chromatography [silicagel 100-200 mesh; elution 0-50% EtOAc in hexane] to afford the desiredproduct (190 mg, 48.74%) as yellow semi solid. LCMS: 378.2 [M+1]⁺.

Step-2: Synthesis of tert-butyl6-((2-cyclopropyl-1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-1,1-dimethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of2-cyclopropyl-1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one.(190 mg, 0.5 mmol, 1.0 eq) in toluene (3.0 mL) was added m-CPBA (245 mg,1.0 mmol, 2.0 eq) and allowed to stir at RT for 60 minutes. Tert-butyl6-amino-1,1-dimethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (150 mg,0.6 mmol, 1.2 eq) and DIPEA (0.4 mL, 2.0 mmol, 4.0 eq) were added andallowed to stir at RT overnight. After completion of reaction, thereaction mixture was diluted with water and extracted with EtOAc (50mL×2). The combined organic layer was washed with water (50 mL) andbrine solution (50 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure to afford crude product, which waspurified by flash chromatography [silica gel 100-200 mesh; elution 0-50%EtOAc in hexane] to afford the desired compound (70 mg, 23.14%) as lightyellow solid. LCMS: 606.32 [M+1]⁺.

Step-3: Synthesis of2-cyclopropyl-6-((1,1-dimethyl-1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:Tert-butyl6-((2-cyclopropyl-1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-1,1-dimethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate(70 mg, 0.11 mmol, 1.0 eq) was dissolved in dioxane (1.5 mL), followedby dropwise addition of 4.0 M-HCl in dioxane (4 mL) and allowed to stirat RT for 2 h. After completion of reaction, the reaction mixture wasfiltered and dried under reduced pressure, crude was given for preppurification to afford the desired compound (2.5 mg, 3.9%) as lightyellow solid. LCMS: 506.4 [M+1]⁺; ¹H NMR (400 MHz, DMSO-d₆, Free base):δ 10.20 (br sbr s, 1H) 8.81 (s, 1H) 8.08-8.20 (m, 2H) 7.98 (d, J=6.58Hz, 1H) 7.58 (br s, 1H) 7.45 (d, J=8.33 Hz, 1H) 7.30 (d, J=7.89 Hz, 1H)3.25 (br s, 1H), 3.14 (br s, 2H) 2.86 (br s, 2H) 1.78 (s, 3H) 1.72 (s,3H) 1.51 (s, 6H) 0.82 (br s, 4H).

Example S-7: Synthesis of1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(Compound No. 1.249)

Step-1: Synthesis of1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:To a stirred solution of2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(287 mg, 1.28 mmol, 1.0 eq) and2-(6-bromo-3-fluoropyridin-2-yl)propan-2-ol (300 mg, 1.28 mmol, 1.0 eq)in dioxane (10 mL) was added potassium carbonate (354.09 mg, 2.56 mmol,2.0 eq) and the resulting mixture was purged with nitrogen for 10 min,followed by addition of copper iodide (48.8 mg, 0.25 mmol, 0.2 eq), andN,N′-dimethylethylenediamine (DMEDA) (0.05 mL, 0.51 mmol, 0.4 eq) andagain purged with nitrogen for 10 min, then stirred at 110° C.overnight. After completion of reaction, the reaction mixture wasdiluted with water and extracted with EtOAc (50 mL×2). Combined organiclayers were washed with water (50 mL) and brine solution (50 mL), driedover anhydrous sodium sulphate and concentrated under reduced pressureto afford crude product, which was purified by flash chromatography toafford the desired compound. LCMS: 378.13 [M+1]⁺.

Step-2: Synthesis of tert-butyl6-((1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(275 mg, 0.73 mmol, 1.0 eq) in toluene (3.0 mL) mL was added m-CPBA (251mg, 1.46 mmol, 2.0 eq) and allowed to stir at RT for 1 h. Tert-butyl6-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (199 mg, 0.80 mmol,1.2 eq) and DIPEA (0.5 mL, 2.92 mmol, 4.0 eq) were added and allowed tostir at RT overnight. After completion of reaction, the reaction mixturewas diluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layer was washed with water (50 mL) and brine solution (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-50% EtOAc in hexane]to afford the desired compound. LCMS: 578.28 [M+1]⁺.

Step-3: Synthesis of1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:Tert-butyl6-((1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate(381 mg, 0.66 mmol, 1.0 eq) was dissolved in dioxane (4 mL), followed bydropwise addition of 4.0 M-HCl (4 mL) and allowed to stir at RT for 1 h.After completion of reaction, the reaction mixture was filtered anddried under reduced pressure to afford the desired compound. LCMS:478.23 [M+1]⁺; ¹H NMR (400 MHz, DMSO-d₆): δ 10.20 (br s, 1H) 8.81 (s,1H) 8.30 (s, 1H) 7.93-8.03 (m, 1H) 7.83 (dd, J=8.33, 2.63 Hz, 1H) 7.56(br s, 1H) 7.40 (d, J=8.33 Hz, 1H) 7.02 (d, J=8.33 Hz, 1H) 4.17-4.21 (m,1H) 3.93 (s, 3H) 3.09 (t, J=5.92 Hz, 3H) 2.76 (t, J=5.48 Hz, 2H) 1.52(s, 6H) 1.32 (d, J=6.58 Hz, 6H).

Example S-8: Synthesis of1-(6-(2-isopropyl-3-oxo-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropane-1-carbonitrile.(Compound No. 1.43)

Step-1: Synthesis of 2-(6-bromopyridin-2-yl)acetonitrile: To a stirredsolution of MeCN (1.4 mL, 27.4 mmol, 3.6 eq) in THF (30 mL) was addedn-BuLi (2.5M in hexane, 10 mL, 25.1 mmol, 3.3 eq) at −78° C. and stirredat −78° C. for 30 min. After stirring for 30 min at −78° C.,2,6-dibromopyridine (1.8 g, 7.6 mmol, 1.0 eq) in THF (10 mL) was added.The reaction mixture was stirred at −78° C. for 45 min. After completionof reaction, the reaction mixture was diluted with water and extractedwith EtOAc (50 mL×2). The combined organic layers were washed with water(50 mL) and brine solution (50 mL), dried over anhydrous sodium sulphateand concentrated under reduced pressure to afford crude product, whichwas purified by flash chromatography [Combiflash, elution-O-30% EtOAc inhexane]to afford the desired compound. LCMS: 197.04 [M+1]⁺.

Step-2: Synthesis of 1-(6-bromopyridin-2-yl)cyclopropane-1-carbonitrile:To a stirred solution of 2-(6-bromopyridin-2-yl)acetonitrile (2.0 g,10.15 mmol, 1.0 eq) in THF (30 mL) was added LiHMDS (22.33 mL, 22.33mmol, 2.2 eq) at −78° C. dropwise under nitrogen atmosphere. Afterstirring for 30 min at −78° C., dibromoethane (2.0 g, 11.16 mmol, 1.1eq) was added and the reaction mixture was stirred at RT overnight.After completion of reaction, the reaction mixture was diluted withammonium chloride (200 mL) and extracted with EtOAc (250 mL×2). Thecombined organic layers were washed with water (250 mL) and brinesolution (250 mL), dried over anhydrous sodium sulphate and concentratedunder reduced pressure to afford crude product, which was purified byflash chromatography to afford the desired compound. LCMS: 224.09[M+1]⁺.

Step-3: Synthesis of1-(6-(2-isopropyl-6-(methylthio)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropane-1-carbonitrile:To a stirred solution of1-(6-bromopyridin-2-yl)cyclopropane-1-carbonitrile (600 mg, 2.67 mmol,1.0 eq) and (602 mg, 2.67 mmol, 1.0 eq) in dioxane (10 mL) was addedpotassium carbonate (800 mg, 5.34 mmol, 2.0 eq) and the resultingmixture was purged with nitrogen for 10 min followed by addition ofcopper iodide (40 mg, 190.4 mmol, 0.2 eq), andN,N′-dimethylethylenediamine (DMEDA) (0.1 mL, 1.06 mmol, 0.4 eq) andagain purged with nitrogen for 10 min, then stirred at 100° C.overnight. After completion of reaction, the reaction mixture wasdiluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layers were washed with water (50 mL) and brine solution (50mL), dried over anhydrous sodium sulphate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography to afford the desired compound. LCMS: 367.2 [M+1]⁺.

Step-4: Synthesis tert-butyl6-((1-(6-(1-cyanocyclopropyl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of1-(6-(2-isopropyl-6-(methylthio)-3-oxo-2,3-dihydro-1H-pyrazolol[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropane-1-carbonitrile(100 mg, 0.28 mmol, 1.0 eq) in toluene (2.0 mL) was added m-CPBA (98.17mg, 0.56 mmol, 2.0 eq) and allowed to stir at RT for 1 h. Tert-butyl6-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (84 mg, 0.34 mmol, 1.5eq) and DIPEA (0.2 mL, 1.42 mmol, 5.0 eq) were added and allowed to stirat RT overnight. After completion of reaction, the reaction mixture wasdiluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layer was washed with water (50 mL) and brine solution (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-70% EtOAc in hexane]to afford the desired compound. LCMS: 567.3 [M+1]⁺.

Step-5: Synthesis of1-(6-(2-isopropyl-3-oxo-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropane-1-carbonitrile:Tert-butyl6-((1-(6-(1-cyanocyclopropyl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate(90 mg, 0.19 mmol, 1.0 eq) was dissolved in dioxane (1 mL), followed bydropwise addition of 4.0 M-HCl (0.5 mL) and allowed to stir at RT for 4h. After completion of reaction, the reaction mixture was filtered anddried under reduced pressure and purified by reverse phase HPLC toafford the desired compound. LCMS: 467.4 [M+1]⁺; ¹H NMR (400 MHz,DMSO-d₆): δ 10.29 (br s, 1H) 8.82 (s, 1H) 8.19 (s, 1H) 8.09-8.16 (m, 1H)7.84 (d, J=8.33 Hz, 1H) 7.63 (br s, 1H) 7.57 (s, 1H) 7.42 (d, J=8.77 Hz,1H) 7.06 (s, 1H) 3.98-4.09 (m, 2H) 3.17 (br s, 4H) 2.85 (br s, 2H)1.82-1.90 (m, 2H) 1.64-1.74 (m, 2H) 1.35 (d, J=6.58 Hz, 6H).

Example S-9: Synthesis of1-(6-(6-(1,1-dimethyl-1,2,3,4-tetrahydroisoquinolin-6-ylamino)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropanecarbonitrile(Compound No. 1.45)

Step-1: Synthesis of tert-butyl6-(1-(6-(1-cyanocyclopropyl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-ylamino)-1,1-dimethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of1-(6-(2-isopropyl-6-(methylthio)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropanecarbonitrile(100 mg, 0.28 mmol, 1.0 eq) in toluene (2.0 mL) was added m-CPBA (138mg, 0.56 mmol, 2.0 eq) and allowed to stir at RT for 1 h. Tert-butyl6-amino-1,1-dimethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (94 mg,0.34 mmol, 1.1 eq) and DIPEA (0.2 mL, 1.4 mmol, 5.0 eq) were added andallowed to stir at RT overnight. After completion of reaction, thereaction mixture was diluted with water and extracted with EtOAc (50mL×2). The combined organic layer was washed with water (50 mL) andbrine solution (50 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure to afford crude product, which waspurified by flash chromatography [silica gel 100-200 mesh; elution 0-50%EtOAc in hexane] to afford the desired compound. LCMS: 595.4 [M+1]⁺.

Step-2: Synthesis of1-(6-(6-(1,1-dimethyl-1,2,3,4-tetrahydroisoquinolin-6-ylamino)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropanecarbonitrile:Tert-butyl6-(1-(6-(1-cyanocyclopropyl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-ylamino)-1,1-dimethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate(80 mg, 0.13 mmol, 1.0 eq) was dissolved in dioxane (0.8 mL), followedby dropwise addition of 4.0 M-HCl (0.8 mL) and allowed to stir at RT for1 h. After completion of reaction, the reaction mixture was filtered andpurified by reverse phase chromatography of afford the desired compound.LCMS: 495.5 [M+1]⁺+; ¹H NMR (400 MHz, DMSO-d₆): δ 10.18 (br s, 1H) 8.80(s, 1H) 8.27 (s, 1H) 8.10-8.13 (m, 1H) 7.83 (d, J=8.33 Hz, 1H) 7.57 (s,1H) 7.51 (br s, 1H) 7.37 (br s, 1H) 7.20 (d, J=8.77 Hz, 1H) 4.09 (d,J=6.58 Hz, 1H) 3.02 (br s, 2H) 2.68 (d, J=9.65 Hz, 2H) 1.85 (d, J=3.51Hz, 2H) 1.71 (d, J=3.07 Hz, 2H) 1.29-1.44 (m, 12H).

Example S-10: Synthesis of 21-(4-(tert-butyl)-5-chlorothiazol-2-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(Compound No. 1.349)

Step-1: Synthesis of 2-bromo-4-(tert-butyl)-5-chlorothiazole: To astirred solution of 2-bromo-4-(tert-butyl)thiazole (1.21 g, 5.49 mmol,1.0 eq) in ACN (20 mL), was added NCS (0.80, 6.04 mmol, 1.1 eq). Thereaction mixture was stirred at RT for 12 h. After completion ofreaction, the reaction mixture was diluted with water (50 mL) and wasextracted with EtOAc (50 mL×2). The combined organic layers were washedwith water (50 mL) and brine solution (50 mL), dried over anhydroussodium sulfate and concentrated under reduced pressure to afford crudeproduct, which was purified by flash chromatography to afford thedesired compound. LCMS: 254.2 [M+2]⁺.

Step-2: Synthesis of1-(4-(tert-butyl)-5-chlorothiazol-2-yl)-2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:To a stirred solution of2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(865 mg, 3.85 mmol, 1.0 eq) and 2-bromo-4-(tert-butyl)-5-chlorothiazole(982 mg, 3.85 mmol, 1.0 eq) in dioxane (12 mL) was added potassiumcarbonate (1.06 g, 7.71 mmol, 2.0 eq) and the resulting mixture waspurged with nitrogen for 10 min, followed by addition of copper iodide(146 mg, 0.77 mmol, 0.2 eq), and N,N′-dimethylethylenediamine (DMEDA)(0.16 mL, 1.54 mmol, 0.4 eq) and again purged with nitrogen for 10 min,then stirred at 90° C. overnight. After completion of reaction, thereaction mixture was diluted with water and extracted with EtOAc (50mL×2). The combined organic layers were washed with water (50 mL) andbrine solution (50 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure to afford crude product, which waspurified by flash chromatography to afford the desired product. LCMS:298.2 [M+1]⁺.

Step-3: Synthesis of tert-butyl7-((1-(4-(tert-butyl)-5-chlorothiazol-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of1-(4-(tert-butyl)-5-chlorothiazol-2-yl)-2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(32 mg, 0.08 mmol, 1.0 eq) in toluene (2.0 mL) was added m-CPBA (28 mg,0.16 mmol, 2.0 eq) and allowed to stir at RT for 1 h. Tert-butyl7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (22 mg, 0.31 mmol, 1.1eq) and Na₂CO₃ (34 mg, 0.32 mmol, 4.0 eq) were added and allowed to stirat RT overnight. After completion of reaction, the reaction mixture wasdiluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layer was washed with water (50 mL) and brine solution (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography to afford the desired compound. LCMS: 598.3 [M+1]⁺.

Step-4: Synthesis of 21-(4-(tert-butyl)-5-chlorothiazol-2-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:Tert-butyl7-((1-(4-(tert-butyl)-5-chlorothiazol-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate(25 mg, 0.04 mmol, 1.0 eq) was dissolved in dioxane (0.3 mL), followedby dropwise addition of 4.0 M-HCl (0.3 mL) and allowed to stir at RT for1 h. After completion of reaction, the reaction mixture was filtered anddried under reduced pressure to afford the desired compound. LCMS: 498.4[M+1]⁺; ¹H NMR (400 MHz, DMSO-d₆): δ 10.64 (br s, 1H) 9.31 (br s, 1H)8.89 (s, 1H) 7.72 (br s, 1H) 7.58 (d, J=8.77 Hz, 1H) 7.23 (d, J=8.33 Hz,1H) 4.54-4.67 (m, 1H) 4.28 (br s, 2H) 2.99 (t, J=5.92 Hz, 2H) 1.54 (d,J=6.58 Hz, 2H) 1.38-1.49 (m, 15H).

Example S-11: Synthesis of1-(6-(2-ethyl-3-oxo-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropane-1-carbonitrile(Compound No. 1.37)

Step-1: Synthesis of1-(6-(2-ethyl-6-(methylthio)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropane-1-carbonitrile:To a stirred solution of 2-ethyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one (200 mg, 0.95 mmol, 1.0 eq) and1-(6-bromopyridin-2-yl)cyclopropane-1-carbonitrile (212 mg, 0.95 mmol,1.0 eq) in dioxane (10 mL) was added potassium carbonate (262.9 mg, 2mmol, 2.0 eq) and the resulting mixture was purged with nitrogen for 10min followed by addition of copper iodide (36.23 mg, 0.19 mmol, 0.2 eq),and N,N′-dimethylethylenediamine (DMEDA) (0.04 mL, 0.381 mmol, 0.4 eq)and again purged with nitrogen for 10 min, then stirred at 130° C.overnight. After completion of reaction, the reaction mixture wasdiluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layer was washed with water (50 mL) and brine solution (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-50% EtOAc in hexane]to afford the desired product (216 mg, 64.43%) as yellow semi solid.LCMS: 353.13 (M+1)⁺.

Step-2: Synthesis of tert-butyl6-((1-(6-(1-cyanocyclopropyl)pyridin-2-yl)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of1-(6-(2-ethyl-6-(methylthio)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropane-1-carbonitrile (108 mg,0.31 mmol, 1.0 eq) in toluene (3.0 mL) was added m-CPBA (145.6 mg, 0.61mmol, 2.0 eq) and allowed to stir at RT for 60 min. Tert-butyl6-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (76.10 mg, 0.31 mmol,1.0 eq) and Na₂CO₃ (129.7 mg, 1.22 mmol, 4.0 eq) were added and allowedto stir at RT overnight. After completion of reaction, the reactionmixture was diluted with water and extracted with EtOAc (50 mL×2). Thecombined organic layer was washed with water (50 mL) and brine solution(50 mL), dried over anhydrous sodium sulfate and concentrated underreduced pressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-50% EtOAc in hexane]to afford the desired compound (121 mg, 71.44%) as light yellow solid.LCMS: 553.26 (M+1)⁺.

Step-3: Synthesis of1-(6-(2-ethyl-3-oxo-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropane-1-carbonitrile:Tert-butyl6-((1-(6-(1-cyanocyclopropyl)pyridin-2-yl)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate(121 mg, 0.22 mmol, 1.0 eq) was dissolved in dioxane (1 mL), followed bydropwise addition of 4.0 M-HCl in dioxane (1.5 mL) and allowed to stirat RT for 2 h. After completion of reaction, the reaction mixture wasfiltered and dried under reduced pressure; and crude was purified byreverse phase chromatography to afford the desired compound (10 mg,10.00%) as an off-white solid. LCMS: 453.21 (M+1)⁺; ¹H NMR (400 MHz,DMSO-d₆): δ 10.27 (br s, 1H) 8.86 (s, 1H) 8.24 (s, 1H) 8.11 (t, J=7.89Hz, 1H) 7.91 (d, J=8.33 Hz, 1H) 7.62 (br s, 1H) 7.53 (d, J=7.45 Hz, 1H)7.39 (br s, 1H) 6.98-7.05 (m, 1H) 3.83-4.05 (m, 4H) 3.10 (br s, 2H) 2.78(br s, 2H) 1.82-1.96 (m, 2H) 1.61-1.74 (m, 2H) 0.96 (t, J=7.02 Hz, 3H).

Example S-12: Synthesis of1-(6-(2-ethyl-3-oxo-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropane-1-carbonitrile(Compound No. 1.38)

Step-1: Synthesis of tert-butyl7-((1-(6-(1-cyanocyclopropyl)pyridin-2-yl)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of2-ethyl-1-(6-(1-methylcyclopropyl)pyridin-2-yl)-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one (108 mg, 0.31 mmol, 1.0 eq) in toluene (3.0 mL)was added m-CPBA (145.6 mg, 0.61 mmol, 2.0 eq) and allowed to stir at RTfor 60 minutes. Tert-butyl7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (76.01 mg, 0.31 mmol,1.0 eq) and Na₂CO₃ (129.7 mg, 1.22 mmol, 4.0 eq) were added and allowedto stir at RT overnight. After completion of reaction, the reactionmixture was diluted with water and extracted with EtOAc (50 mL×2). Thecombined organic layer was washed with water (50 mL) and brine solution(50 mL), dried over anhydrous sodium sulfate and concentrated underreduced pressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-50% EtOAc in hexane]to afford the desired compound, (119 mg, 70.2%) as light yellow solid.LCMS: 553.26 (M+1)⁺.

Step-2: Synthesis of1-(6-(2-ethyl-3-oxo-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropane-1-carbonitriledihydrochloride: Tert-butyl7-((1-(6-(1-cyanocyclopropyl)pyridin-2-yl)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate(119 mg, 0.21 mmol, 1.0 eq) was dissolved in dioxane (1 mL), followed bydropwise addition of 4.0 M-HCl in dioxane (1.5 mL) and allowed to stirat RT for 2 h. After completion of reaction, the reaction mixture wasfiltered and dried under reduced pressure and purified product wasobtained by reverse phase chromatography (24 mg, 21.2%) as white solid.LCMS: 453.21 (M+1)⁺; ¹H NMR (400 MHz, DMSO-d₆): δ 10.39 (br s, 1H) 9.26(br s, 1H) 8.88 (s, 1H) 8.15 (t, J=7.6 Hz, 1H) 7.93 (d, J=7.8 Hz, 1H)7.68 (br s, 1H) 7.43-7.56 (m, 2H) 7.20 (d, J=8.3 Hz, 1H) 4.26 (br s, 2H)3.81-3.98 (m, 2H) 3.38 (d, J=5.7 Hz, 2H) 2.97 (t, J=5.9 Hz, 2H)1.82-1.92 (m, 2H) 1.65-1.74 (m, 2H) 0.97 (t, J=7.0 Hz, 3H).

Example S-13: Synthesis of1-(6-(2-isopropyl-3-oxo-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropane-1-carbonitrile(Compound No. 1.44)

Step-1: Synthesis of1-(6-(2-isopropyl-6-(methylthio)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropane-1-carbonitrile:To a stirred solution of2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(100 mg, 0.45 mmol, 1.0 eq) and1-(6-bromopyridin-2-yl)cyclopropane-1-carbonitrile (100 mg, 0.45 mmol,1.0 eq) in dioxane (6 mL) was added potassium carbonate (123 mg, 0.89mmol, 2.0 eq) and the resulting mixture was purged with nitrogen for 10min followed by addition of copper iodide (17 mg, 0.089 mmol, 0.2 eq),and N,N′-dimethylethylenediamine (DMEDA) (0.02 mL, 0.178 mmol, 0.4 eq)and again purged with nitrogen for 10 min, then stirred at 130° C.overnight. After completion of reaction, the reaction mixture wasdiluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layer was washed with water (50 mL) and brine solution (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-40% EtOAc in hexane]to afford the desired product (102 mg, 62.4%) as colorless liquid. LCMS:367.13 (M+1)⁺.

Step-2: Synthesis of tert-butyl7-((1-(6-(1-cyanocyclopropyl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of1-(6-(2-isopropyl-6-(methylthio)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropane-1-carbonitrile(102 mg, 0.28 mmol, 1.0 eq) in toluene (3.0 mL) was added m-CPBA (132mg, 0.56 mmol, 2.0 eq) and allowed to stir at RT for 60 minutes.Tert-butyl 6-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (75.9 mg,0.31 mmol, 1.0 eq) and Na₂CO₃ (117.8 mg, 1.11 mmol, 4.0 eq) were addedand allowed to stir at RT overnight. After completion of reaction, thereaction mixture was diluted with water and extracted with EtOAc (50mL×2). The combined organic layer was washed with water (50 mL) andbrine solution (50 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure to afford crude product, which waspurified by flash chromatography [silica gel 100-200 mesh; elution 0-50%EtOAc in hexane] to afford the desired compound (90 mg, 57.0%) as anoff-white solid. LCMS: 567.28 (M+1)⁺.

Step-3: Synthesis of1-(6-(2-isopropyl-3-oxo-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropane-1-carbonitrile:Tert-butyl7-((1-(6-(1-cyanocyclopropyl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate(90 mg, 0.15 mmol, 1.0 eq) was dissolved in dioxane (1 mL), followed bydropwise addition of 4.0 M-HCl in dioxane (2 mL) and allowed to stir atRT for 2 h. After completion of reaction, the reaction mixture wasfiltered and dried under reduced pressure, purified product was obtainedby reverse phase chromatography (40 mg, 46.7%) as an off-white solid.LCMS: −467.22 (M+1)⁺; ¹H NMR (400 MHz, DMSO-d₆): δ 10.35 (br s, 1H) 9.11(br s, 2H) 8.83 (s, 1H) 8.08-8.20 (m, 1H) 7.87 (s, 1H) 7.65 (br s, 1H)7.51 (d, J=8.3 Hz, 1H) 7.55 (d, J=7.5 Hz, 1H) 7.19 (d, J=8.7 Hz, 1H)4.26 (br s, 2H) 4.09 (m, 1H) 3.39 (t, 2H) 2.96 (t, 2H) 1.87 (d, J=3.5Hz, 2H) 1.71 (d, J=3.1 Hz, 2H) 1.36 (d, J=6.5 Hz, 6H).

Example S-14: Synthesis of1-(4-(2-isopropyl-3-oxo-6-(1,2,3,4-tetrahydroisoquinolin-6-ylamino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropanecarbonitrile(Compound No. 1.67)

Step-1: Synthesis of 1-(4-bromopyridin-2-yl)cyclopropanecarbonitrile: Toa stirred solution of cyclopropanecarbonitrile (1.0 g, 14.5 mmol, 1.0eq), in toluene (10 mL) was added LiHMDS (16 mL, 15.9 mmol, 1.1 eq), at0° C. & the reaction mixture was stirred for 1 h.2-Fluoro-4-bromopyridine in toluene (5 mL) (2.56 g, 14.4 mmol, 1.0 eq)was added dropwise & stirred for 18 h. After completion of reaction, thereaction mixture was diluted with saturated NH₄Cl solution and extractedwith diethyl ether (50 mL×2). The combined organic layer was washed withwater (50 mL) and brine solution (50 mL), dried over anhydrous sodiumsulfate and concentrated under reduced pressure to afford crude product,which was purified by flash chromatography [silica gel 100-200 mesh;elution 0-50% EtOAc in hexane] to afford the desired compound (2.1 g,65.0%) as colorless solid. LCMS: 222.9 (M+1)⁺.

Step-2: Synthesis of1-(4-(2-isopropyl-6-(methylthio)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropanecarbonitrile:To a stirred solution of2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(224 mg, 1.0 mmol, 1.0 eq) and1-(4-bromopyridin-2-yl)cyclopropanecarbonitrile (223 mg, 1.0 mmol, 1.0eq) in f dioxane (10 mL) were added potassium carbonate (276 mg, 2.0mmol, 2.0 eq) and the resulting mixture was purged with nitrogen for 10min followed by addition of copper iodide (38 mg, 0.2 mmol, 0.2 eq), andN,N′-dimethylethylenediamine (DMEDA) (0.05 mL, 0.4 mmol, 0.4 eq) andagain purged with nitrogen for 10 min, then stirred at 90° C. for 48 h.After completion of reaction, the reaction mixture was diluted withwater and extracted with EtOAc (50 mL×2). The combined organic layerswere washed with water (50 mL) and brine solution (50 mL), dried overanhydrous sodium sulphate and concentrated under reduced pressure toafford crude product, which was purified by flash chromatography toafford the desired compound (50 mg, 13.6%). LCMS: 367.2 (M+1)+

Step-3: Synthesis of tert-butyl6-(1-(2-(1-cyanocyclopropyl)pyridin-4-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-ylamino)-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of1-(4-(2-isopropyl-6-(methylthio)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropanecarbonitrile(50 mg, 0.14 mmol, 1.0 eq) in toluene (2.0 mL) was added m-CPBA (48 mg,0.28 mmol, 2.0 eq) and allowed to stir at RT for 1 h. Tert-butyl6-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (38 mg, 0.15 mmol, 1.1eq) and Na₂CO₃ (58 mg, 0.55 mmol, 4.0 eq) were added and allowed to stirat RT overnight. After completion of reaction, the reaction mixture wasdiluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layer was washed with water (50 mL) and brine solution (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-50% EtOAc in hexane]to afford the desired compound (40 mg, 51.9%) as an off-white solid.LCMS: 567.4 (M+1)⁺.

Step-4: Synthesis of1-(4-(2-isopropyl-3-oxo-6-(1,2,3,4-tetrahydroisoquinolin-6-ylamino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropanecarbonitrile:Tert-butyl6-(1-(2-(1-cyanocyclopropyl)pyridin-4-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-ylamino)-3,4-dihydroisoquinoline-2(1H)-carboxylate(40 mg, 0.07 mmol, 1.0 eq) was dissolved in DCM (5 mL), followed bydropwise addition of Trifluoro acetic acid (0.51 mL) and allowed to stirat RT for 1 h. After completion of reaction, the reaction mixture wasdried under reduced pressure and purified product was obtained byreverse phase purification (20 mg, 55.4%) as white solid. LCMS: 467.5(M+1)⁺; ¹H NMR (400 MHz, DMSO-d₆): δ 10.37 (br s, 1H) 8.85 (s, 1H) 8.65(d, J=5.2 Hz, 1H) 7.56-7.65 (m, 2H) 7.42-7.56 (m, 2H) 7.06 (d, J=8.3 Hz,1H) 3.87-4.03 (m, 3H) 3.08 (d, J=5.7 Hz, 2H) 2.78 (br s, 2H) 1.84-1.95(m, 2H) 1.65-1.79 (m, 2H) 1.37 (d, J=7.0 Hz, 6H).

Example S-15: Synthesis of Synthesis of2-ethyl-1-(6-(1-(fluoromethyl)cyclopropyl)pyridin-2-yl)-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one (Compound No. 1.86)

Step-1: Synthesis of2-ethyl-1-(6-(1-(fluoromethyl)cyclopropyl)pyridin-2-yl)-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:To a stirred solution of2-ethyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(172.6 mg, 0.82 mmol, 1.0 eq) and2-bromo-6-(1-(fluoromethyl)cyclopropyl)pyridine (189.0 mg, 0.82 mmol,1.0 eq) in dioxane (10 mL) was added potassium carbonate (227 mg, 1.64mmol, 2.0 eq) and the resulting mixture was purged with nitrogen for 10min followed by addition of copper iodide (31 mg, 0.16 mmol, 0.2 eq),and N,N′-dimethylethylenediamine (DMEDA) (0.03 mL, 0.33 mmol, 0.4 eq)and again purged with nitrogen for 10 min, then stirred at 130° C.overnight. After completion of reaction, the reaction mixture wasdiluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layer was washed with water (50 mL) and brine solution (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-50% EtOAc in hexane]to afford the desired product (213 mg, 72.8%) as brown semi-solid. LCMS:360.12 (M+1)⁺.

Step-2: Synthesis tert-butyl7-((2-ethyl-1-(6-(1-(fluoromethyl)cyclopropyl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of2-ethyl-1-(6-(1-(fluoromethyl)cyclopropyl)pyridin-2-yl)-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(213 mg, 0.82 mmol, 1.0 eq) in toluene (2.0 mL) was added m-CPBA (405mg, 1.64 mmol, 2.0 eq) and allowed to stir at RT for 60 minutes.Tert-butyl 7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (204 mg,0.82 mmol, 1.0 eq) and Na₂CO₃ (348 mg, 3.28 mmol, 4.0 eq) were added andallowed to stir at RT overnight. After completion of reaction, thereaction mixture was diluted with water and extracted with EtOAc (50mL×2). The combined organic layer was washed with water (50 mL) andbrine solution (50 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure to afford crude product, which waspurified by flash chromatography [silica gel 100-200 mesh; elution 0-50%EtOAc in hexane] to afford the desired compound (60 mg, 18.1%) as lightyellow solid. LCMS: 560.27 (M+1)⁺.

Step-3: Synthesis of2-ethyl-1-(6-(1-(fluoromethyl)cyclopropyl)pyridin-2-yl)-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:Tert-butyl7-((2-ethyl-1-(6-(1-(fluoromethyl)cyclopropyl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate(60 mg, 0.11 mmol, 1.0 eq) was dissolved in dioxane (1 mL), followed bydropwise addition of 2.0 M-HCl/Dioxane (2.0 mL) and allowed to stir atRT for 2 h. After completion of reaction, the reaction mixture wasfiltered and dried under reduced pressure and the purified product wasobtained by reverse phase chromatography (8 mg, 16.2%) as a white solid.LCMS: 460.22 (M+1)⁺; ¹H NMR (400 MHz, DMSO-d₆): δ10.25 (br s, 1H) 8.87(s, 1H) 8.26 (s, 1H), 8.15 (t, J=7.7 Hz, 1H) 8.00 (d, J=7.9 Hz, 1H) 7.62(br s, 1H) 7.58 (br s, 1H) 7.51 (d, J=7.5 Hz, 1H) 7.05 (d, J=7.8 Hz, 1H)3.99-4.12 (m, 2H) 3.92 (s, 2H) 2.97-3.10 (m, 2H) 2.33 (br s, 2H) 2.00(br s, 2H) 1.81 (d, J=8.7 Hz, 2H) 1.00 (t, J=7.2 Hz, 3H).

Example S-16: Synthesis of2-ethyl-1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(Compound No. 1.91)

Step-1: Synthesis of 2-bromo-6-(1-(fluoromethyl)cyclopropyl)pyridine: Toa stirred solution of (1-(6-bromopyridin-2-yl)cyclopropyl)methanol (1 g,4.38 mmol, 1.0 eq) in DCM (40 mL) at −78° C. followed by addition ofDAST (0.9 mL, 6.57 mmol, 1.5 eq) and allowed to stir at RT overnight.After completion of reaction, the reaction mixture was basified withNaHCO₃ (20 mL) at −78° C. and extracted with DCM (100 mL×2). Thecombined organic layer was washed with water (50 mL) and brine solution(50 mL), dried over anhydrous sodium sulfate and concentrated underreduced pressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-40% EtOAc in hexane]to afford the desired compound as gummy material. LCMS: 229.99 (M+1)⁺.

Step-2: Synthesis of1-(6-(1-(fluoromethyl)cyclopropyl)pyridin-2-yl)-2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:To a stirred solution of 2-bromo-6-(1-(fluoromethyl)cyclopropyl)pyridine(189 mg, 0.82 mmol, 1.0 eq) and2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one (184.23 mg, 0.82 mmol, 1.0 eq) in dioxane (10 mL)was added potassium carbonate (227 mg, 1.64 mmol, 2.0 eq) and theresulting mixture was purged with nitrogen for 10 min followed byaddition of copper iodide (31 mg, 0.16 mmol, 0.2 eq), andN,N′-dimethylethylenediamine (DMEDA) (0.03 mL, 0.33 mmol, 0.4 eq) andagain purged with nitrogen for 10 min, then stirred at 130° C.overnight. After completion of reaction, the reaction mixture wasdiluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layer was washed with water (50 mL) and brine solution (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-50% EtOAc in hexane]to afford the desired product (133 mg, 43.35%) as brown semi-solid.LCMS: 374.14 (M+1)⁺.

Step-3: Synthesis of tert-butyl6-((1-(6-(1-(fluoromethyl)cyclopropyl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of1-(6-(1-(fluoromethyl)cyclopropyl)pyridin-2-yl)-2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(133 mg, 0.36 mmol, 1.0 eq) in toluene (2.0 mL) was added m-CPBA (170mg, 0.712 mmol, 2.0 eq) and allowed to stir at RT for 60 minutes.Tert-butyl 6-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (88.4 mg,0.36 mmol, 1.0 eq) and Na₂CO₃ (151 mg, 1.42 mmol, 4.0 eq) were added andallowed to stir at RT overnight. After completion of reaction, thereaction mixture was diluted with water and extracted with EtOAc (50mL×2). The combined organic layer was washed with water (50 mL) andbrine solution (50 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure to afford crude product, which waspurified by flash chromatography [silica gel 100-200 mesh; elution 0-50%EtOAc in hexane] to afford the desired compound (61 mg, 30.04%) as lightyellow solid. LCMS: 574.29 (M+1)⁺.

Step-4: Synthesis of1-(6-(1-(fluoromethyl)cyclopropyl)pyridin-2-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:Tert-butyl6-((1-(6-(1-(fluoromethyl)cyclopropyl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate(61 mg, 0.106 mmol, 1.0 eq) was dissolved in dioxane (1 mL) followed bydropwise addition of 2.0 M-HCl in diethylether (2.0 mL) and allowed tostir at RT for 2 h. After completion of reaction, the reaction mixturewas filtered and dried under reduced pressure and purified product wasobtained by reverse phase chromatography (10 mg, 18.13%) as white solid.LCMS: 474.23 (M+1)⁺; ¹H NMR (400 MHz, DMSO-d₆): δ 10.24 (br s, 1H) 8.83(s, 1H) 8.23 (br s, 1H) 8.14 (t, J=8.1 Hz, 1H) 7.91 (d, J=7.8 Hz, 1H)7.62 (br s, 1H) 7.52 (d, J=7.5 Hz, 1H) 7.40 (d, J=7.8 Hz, 1H) 7.03 (d,J=7.8 Hz, 1H) 4.14-4.27 (m, 1H) 3.96 (br s, 2H) 3.12 (br s, 2H) 2.79 (brs, 2H) 2.67 (br s, 2H) 2.59 (d, J=10.1 Hz, 1H) 2.01 (br s, 2H) 1.81 (dd,J=16.8, 8.1 Hz, 1H) 1.38 (d, J=7.0 Hz, 6H).

Example S-17: Synthesis of2-ethyl-1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(Compound No. 1.188)

Step-1: Synthesis of2-ethyl-1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:To a stirred solution of2-ethyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(244 mg, 1.65 mmol, 1.0 eq) and6-bromo-3-fluoro-2-(2-fluoropropan-2-yl)pyridine (275 mg, 1.65 mmol, 1.0eq) in dioxane (2.0 mL) was added potassium carbonate (456 mg, 3.3 mmol,2.0 eq) and the resulting mixture was purged with nitrogen for 10 minfollowed by addition of copper iodide (62.8 mg, 0.33 mmol, 0.2 eq), andN,N′-dimethylethylenediamine (DMEDA) (0.06 mL, 0.66 mmol, 0.4 eq) andagain purged with nitrogen for 10 min, then stirred at 130° Cbovernight. After completion of reaction, the reaction mixture wasdiluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layer was washed with water (50 mL) and brine solution (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-50% EtOAc in hexane]to afford the desired product (234 mg, 55.2%) as yellow semi solid.LCMS: 366.11 (M+1)⁺.

Step-2: Synthesis of tert-butyl7-((2-ethyl-1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of2-ethyl-1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(100 mg, 0.27 mmol, 1.0 eq) in toluene (3.0 mL) was added m-CPBA (130.5mg, 0.55 mmol, 2.0 eq) and allowed to stir at RT for 60 minutes.Tert-butyl 7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (74.7 mg,0.30 mmol, 1.2 eq) and Na₂CO₃ (116.2 mg, 1.09 mmol, 4.0 eq) were addedand allowed to stir at RT overnight. After completion of reaction, thereaction mixture was diluted with water and extracted with EtOAc (50mL×2). The combined organic layer was washed with water (50 mL) andbrine solution (50 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure to afford crude product, which waspurified by flash chromatography [silica gel 100-200 mesh; elution 0-50%EtOAc in hexane] to afford the desired compound (145 mg, 94.2%) as lightbrown solid. LCMS: 566.26 (M+1)⁺.

Step-3: Synthesis of2-ethyl-1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:Tert-butyl7-((2-ethyl-1-(5-fluoro-6-(2-fluoropropan-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate(145 mg, 0.25 mmol, 1.0 eq) was dissolved in dioxane (1 mL), followed bydropwise addition of 4.0 M-HCl in dioxane (1 mL) and allowed to stir atRT for 2 h. After completion of reaction, the reaction mixture wasfiltered and dried under reduced pressure and the purified product wasobtained by reverse phase chromatography (8 mg, 6.7%) as an off-whitesolid. LCMS 466.21 (M+1)⁺; ¹H NMR (400 MHz, DMSO-d₆): δ ppm 10.28 (br s,1H) 8.86 (s, 1H) 7.99-8.18 (m, 2H) 7.56 (br s, 1H) 7.33-7.42 (m, 1H)7.09 (d, J=8.7 Hz, 1H) 4.00 (br s, 4H) 3.11 (br s, 2H) 2.76 (br s, 2H),1.78 (s, 3H) 1.72 (s, 3H) 0.98 (t, J=7.2 Hz, 3H).

Example S-18: Synthesis of1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(Compound No. 1.250)

Step-1: Synthesis of tert-butyl7-((1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(70 mg, 0.18 mmol, 1.0 eq) in toluene (4.0 mL) was added m-CPBA (88 mg,0.37 mmol, 2.0 eq) and allowed to stir at RT for 60 minutes. Tert-butyl7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (46 mg, 0.18 mmol, 1.0eq) and Na₂CO₃ (78.4 mg, 0.74 mmol, 4.0 eq) were added and allowed tostir at RTovernight. After completion of reaction, the reaction mixturewas diluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layer was washed with water (50 mL) and brine solution (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-50% EtOAc in hexane]to afford the desired compound (99 mg, 92.4%) as an off-white solid.LCMS: 578.28 (M+1)⁺.

Step-2: Synthesis of1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:Tert-butyl7-((1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate(99 mg, 0.17 mmol, 1.0 eq) was dissolved in dioxane (1 mL), followed bydropwise addition of 4.0 M-HCl in dioxane (1.5 mL) and allowed to stirat RT for 2 h. After completion of reaction, the reaction mixture wasfiltered and dried under reduced pressure and purified product wasobtained by reverse phase chromatography (51 mg, 51.3%) as white solid.LCMS: 478.25 (M+1)⁺; ¹H NMR (400 MHz, DMSO-d₆): o 10.33 (br s, 1H) 9.18(br s, 2H) 8.84 (s, 1H) 7.96-8.08 (m, 1H) 7.88 (d, J=6.6 Hz, 1H) 7.67(br s, 1H) 7.48 (d, J=7.5 Hz, 1H) 7.17 (d, J=8.7 Hz, 1H) 4.09-4.33 (m,4H) 3.37 (br s, 2H) 2.95 (br s, 2H) 1.42-1.58 (m, 6H) 1.33 (d, J=6.6 Hz,6H).

Example S-19: Synthesis of2-(5-fluoro-6-(2-isopropyl-3-oxo-6-(1,2,3,4-tetrahydroisoquinolin-6-ylamino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)-2-methylpropanenitrile(Compound No. 1.321)

Step-1: Synthesis of 2-(6-bromopyridin-2-yl)-2-cyanopropan-1-ylium: To astirred solution of isobutyronitrile (1.0 g, 14.46 mmol, 1 eq), intoluene (10 mL) was added LiHMDS (17.4 mL, 17.35 mmol, 1.2 eq), at 0° C.& the reaction mixture was stirred for 1 h. 2,6-dibromo-3-fluoropyridine in toluene (5 mL), (3.68 g, 14.46 mmol, 1.0eq) was added dropwise & stirred for 18 h. After completion of reaction,the reaction mixture was diluted with saturated NH₄Cl solution andextracted with diethyl ether (50 mL×2). The combined organic layer waswashed with water (50 mL) and brine solution (50 mL), dried overanhydrous sodium sulfate and concentrated under reduced pressure toafford crude product, which was purified by flash chromatography [silicagel 100-200 mesh; elution 0-50% EtOAc in hexane] to afford the desiredcompound (2.0 g, 57.1%) as colorless liquid. LCMS: 242.9 (M+1)⁺.

Step-2: Synthesis of2-(3-fluoro-6-(2-isopropyl-6-(methylthio)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)-2-methylpropanenitrile:To a stirred solution of2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(224 mg, 1.0 mmol, 1.0 eq) and2-(6-bromo-5-fluoropyridin-2-yl)-2-methylpropanenitrile (243 mg, 1.0mmol, 1.0 eq) in dioxane (10 mL) were added potassium carbonate (276 mg,2.0 mmol, 2.0 eq) and the resulting mixture was purged with nitrogen for10 min followed by addition of copper iodide (38 mg, 0.2 mmol, 0.2 eq),and N,N′-dimethylethylenediamine (DMEDA) (0.05 mL, 0.4 mmol, 0.4 eq) andagain purged with nitrogen for 10 min, then stirred at 90° C. overnight.After completion of reaction, the reaction mixture was diluted withwater and extracted with EtOAc (50 mL×2). The combined organic layerswere washed with water (50 mL) and brine solution (50 mL), dried overanhydrous sodium sulphate and concentrated under reduced pressure toafford crude product, which was purified by flash chromatography toafford the desired compound (250 mg, 64.7%). LCMS: 387.2 (M+1)⁺.

Step-3: Synthesis of tert-butyl6-(1-(6-(2-cyanopropan-2-yl)-3-fluoropyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-ylamino)-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a2-(3-fluoro-6-(2-isopropyl-6-(methylthio)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)-2-methylpropanenitrile(75 mg, 0.19 mmol, 1.0 eq) in toluene (3.0 mL) was added m-CPBA (93 mg,0.39 mmol, 2.0 eq) and allowed to stir at RT for 1 h. Tert-butyl6-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (51 mg, 0.21 mmol,1.05 eq) and Na₂CO₃ (83 mg, 0.78 mmol, 4.0 eq) were added and allowed tostir at RT overnight. After completion of reaction, the reaction mixturewas diluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layer was washed with water (50 mL) and brine solution (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-50% EtOAc in hexane]to afford the desired compound (70 mg, 61.9%) as an off-white solid.LCMS: 587.4 (M+1)⁺.

Step-4: Synthesis of2-(5-fluoro-6-(2-isopropyl-3-oxo-6-(1,2,3,4-tetrahydroisoquinolin-6-ylamino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)-2-methylpropanenitrile:Tert-butyl6-(1-(6-(2-cyanopropan-2-yl)-3-fluoropyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-ylamino)-3,4-dihydroisoquinoline-2(1H)-carboxylate(70 mg, 0.12 mmol, 1.0 eq) was dissolved in dioxane (1 mL), followed bydropwise addition of 4.0 M-HCl in dioxane (1 mL) and allowed to stir atRT for 1 h. After completion of reaction, the reaction mixture wasfiltered and dried under reduced pressure to afford the desired compound(40 mg, 68.9%) as white solid. LCMS: 487.4 (M+1)⁺; ¹H NMR (400 MHz,DMSO-d₆): δ 10.34 (br s, 1H) 9.31 (br s, 1H) 8.83 (s, 1H) 8.13-8.22 (m,1H) 8.01 (dd, J=8.7, 3.1 Hz, 1H) 7.59-7.68 (m, 1H) 7.46 (m, J=8.77 Hz,1H) 7.15 (m, J=8.7 Hz, 1H) 4.09-4.25 (m, 3H) 3.35 (br s, 2H) 2.98 (t,J=5.9 Hz, 2H) 1.73 (s, 6H) 1.34 (d, J=6.5 Hz, 6H).

Example S-20: Synthesis of2-(5-fluoro-6-(2-isopropyl-3-oxo-6-(1,2,3,4-tetrahydroisoquinolin-7-ylamino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)-2-methylpropanenitrile(Compound No. 1.322)

Step-1: Synthesis of tert-butyl7-(1-(6-(2-cyanopropan-2-yl)-3-fluoropyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-ylamino)-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of2-(3-fluoro-6-(2-isopropyl-6-(methylthio)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)-2-methylpropanenitrile(75 mg, 0.19 mmol, 1.0 eq) in toluene (3.0 mL) was added m-CPBA (93 mg,0.39 mmol, 2.0 eq) and allowed to stir at RT for 1 h. Tert-butyl7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (51 mg, 0.21 mmol,1.05 eq) and Na₂CO₃ (83 mg, 0.78 mmol, 4.0 eq) were added and allowed tostir at RT overnight. After completion of reaction, the reaction mixturewas diluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layer was washed with water (50 mL) and brine solution (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-50% EtOAc in hexane]to afford the desired compound (80 mg, 71.4%) as an off-white solid.LCMS: 587.4 (M+1)⁺.

Step-2: Synthesis of2-(5-fluoro-6-(2-isopropyl-3-oxo-6-(1,2,3,4-tetrahydroisoquinolin-7-ylamino)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)-2-methylpropanenitrile:Tert-butyl7-(1-(6-(2-cyanopropan-2-yl)-3-fluoropyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-ylamino)-3,4-dihydroisoquinoline-2(1H)-carboxylate(70 mg, 0.12 mmol, 1.0 eq) was dissolved in dioxane (1 mL), followed bydropwise addition of 4.0 M-HCl in dioxane (1 mL) and allowed to stir atRT for 1 h. After completion of reaction, the reaction mixture wasfiltered and dried under reduced pressure to afford the desired compound(30 mg, 47.3%) as white solid. LCMS: 487.5 (M+1)⁺; ¹H NMR (400 MHz,DMSO-d₆): δ 10.25 (br s, 1H) 8.82 (s, 1H) 8.27 (br s, 1H) 8.16 (d,J=10.1 Hz, 1H) 8.01 (dd, J=8.7, 3.1 Hz, 1H) 7.50 (br s, 1H) 7.40 (d,J=8.3 Hz, 1H) 7.07 (d, J=8.3 Hz, 1H) 4.18 (dt, J=13.8, 6.6 Hz, 1H) 3.95(br s, 2H) 3.07 (br s, 2H) 2.73 (br s, 2H) 1.68-1.83 (m, 6H) 1.36 (d,J=6.5 Hz, 6H).

Example S-21: Synthesis of2-ethyl-1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(Compound No. 1.262)

Step-1: Synthesis of2-ethyl-1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:To a stirred solution of2-ethyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(359 mg, 1.71 mmol, 1.0 eq) and2-(6-bromo-3-fluoropyridin-2-yl)propan-2-ol (400 mg, 1.71 mmol, 1.0 eq)in dioxane (10 mL) was added potassium carbonate (472 mg, 3.42 mmol, 2.0eq) and the resulting mixture was purged with nitrogen for 10 minfollowed by addition of copper iodide (65 mg, 0.34 mmol, 0.2 eq), andN,N′-dimethylethylenediamine (DMEDA) (0.03 mL, 0.684 mmol, 0.4 eq) andagain purged with nitrogen for 10 min, then stirred at 130° C.overnight. After completion of the reaction, the reaction mixture wasdiluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layer was washed with water (50 mL) and brine solution (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-50% EtOAc in hexane]to afford the desired product as brown liquid. LCMS: 364.12 (M+1)⁺.

Step-2: Synthesis of tert-butyl7-((2-ethyl-1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of2-ethyl-1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one (100 mg, 0.27 mmol, 1.0 eq) in toluene (5.0 mL)was added m-CPBA (95 mg, 0.55 mmol, 2.0 eq) and allowed to stir at RTfor 60 minutes. Tert-butyl7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (75 mg, 0.30 mmol, 1.2eq) and Na₂CO₃ (93 mg, 1.09 mmol, 4.0 eq) were added and allowed to stirat RT overnight. After completion of reaction, the reaction mixture wasdiluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layer was washed with water (50 mL) and brine solution (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-50% EtOAc in hexane]to afford the desired compound (150 mg, 96.7%) as light brown solid.LCMS: 564.27 (M+1)⁺.

Step-3: Synthesis of2-ethyl-1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-((1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:Tert-butyl7-((2-ethyl-1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate(150 mg, 0.26 mmol, 1.0 eq) was dissolved in ether (1 mL), followed bydropwise addition of 2.0 M Ether in HCl (10 mL) and allowed to stir atRT for 2 h. After completion of reaction, the reaction mixture wasfiltered and dried under reduced pressure and the purified product wasobtained by reverse phase purification (70 mg, 46.7%) as an off-whitesolid. LCMS: 464.21 (M+1)⁺; ¹H NMR (400 MHz, DMSO-d₆): δ 10.33 (br s,1H) 9.08 (br s, 2H) 8.86 (s, 1H) 7.89-8.09 (m, 2H) 7.70 (br s, 1H) 7.47(d, J=7.5 Hz, 1H) 7.18 (d, J=8.3 Hz, 1H) 4.26 (br s, 2H) 4.00 (d, J=7.4Hz, 2H) 3.36 (br s, 3H) 2.94 (br s, 2H) 1.51 (s, 6H) 0.96 (t, J=7.0 Hz,3H).

Example S-22: Synthesis of6-((1,1-dimethyl-1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(Compound No. 1.251)

Step-1: Synthesis of tert-butyl6-((1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-1,1-dimethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(200 mg, 0.52 mmol, 1.0 eq) in toluene (4.0 mL) was added m-CPBA (231mg, 1.04 mmol, 2.0 eq) and allowed to stir at RT for 60 minutes.Tert-butyl6-amino-1,1-dimethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (146 mg,0.52 mmol, 1.0 eq) and Na₂CO₃ (220 mg, 2.08 mmol, 4.0 eq) were added andallowed to stir at RT overnight. After completion of reaction, thereaction mixture was diluted with water and extracted with EtOAc (50mL×2). The combined organic layer was washed with water (50 mL) andbrine solution (50 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure to afford crude product, which waspurified by flash chromatography [silica gel 100-200 mesh; elution 0-50%EtOAc in hexane] to afford the desired compound, (120 mg, 37.4%) as anoff-white solid. LCMS: 606.28 (M+1)⁺.

Step-2: Synthesis of6-((1,1-dimethyl-1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:Tert-butyl6-((1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-1,1-dimethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate(92 mg, 0.15 mmol, 1.0 eq) was dissolved in dioxane (1 mL), followed bydropwise addition of 4.0 M-HCl in dioxane (1.5 mL) and allowed to stirat RT for 2 h. After completion of reaction, the reaction mixture wasfiltered and dried under reduced pressure and the purified product wasobtained by reverse phase purification (21 mg, 27.3%) as white solid.LCMS: 506.6 (M+1)⁺; ¹H NMR (400 MHz, DMSO-d₆): δ 10.29 (br s, 1H) 8.83(s, 1H) 8.02 (d, J=10.1 Hz, 1H) 7.86 (d, J=6.1 Hz, 1H) 7.64 (br s, 1H)7.49 (d, J=8.7 Hz, 1H) 7.33 (d, J=8.7 Hz, 1H) 4.13-4.25 (m, 2H) 3.40 (brs, 2H) 2.98 (br s, 2H) 1.61 (s, 6H) 1.52 (s, 6H) 1.33 (d, J=6.58 Hz,6H).

Example S-23: Synthesis of6-((2′,3′-dihydro-1′H-spiro[cyclopropane-1,4′-isoquinolin]-7′-yl)amino)-1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(Compound No. 1.254)

Step-1: Synthesis of tert-butyl7′-((1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-1′H-spiro[cyclopropane-1,4′-isoquinoline]-2′(3′H)-carboxylate:To a stirred solution of1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(200 mg, 0.52 mmol, 1.0 eq) in toluene (4.0 mL) was added m-CPBA (249mg, 1.05 mmol, 2.0 eq) and allowed to stir at RT for 60 minutes.Tert-butyl7′-amino-1′H-spiro[cyclopropane-1,4′-isoquinoline]-2′(3′H)-carboxylate(146 mg, 0.52 mmol, 1.0 eq) and Na₂CO₃ (220 mg, 2.08 mmol, 4.0 eq) wereadded and allowed to stir at RT overnight. After completion of reaction,the reaction mixture was diluted with water and extracted with EtOAc (50mL×2). The combined organic layer was washed with water (50 mL) andbrine solution (50 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure to afford crude product, which waspurified by flash chromatography [silica gel 100-200 mesh; elution 0-50%EtOAc in hexane] to afford the desired compound as an off-white solid.LCMS: 604.28 (M+1)⁺.

Step-2: Synthesis of6-((2′,3′-dihydro-1′H-spiro[cyclopropane-1,4′-isoquinolin]-7′-yl)amino)-1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-onedihydrochloride: Tert-butyl7′-((1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-1′H-spiro[cyclopropane-1,4′-isoquinoline]-2′(3′H)-carboxylate (80 mg, 0.13 mmol,1.0 eq) was dissolved in dioxane (1 mL), followed by dropwise additionof 4.0 M-HCl in dioxane (1.5 mL) and allowed to stir at RT for 2 h.After completion of reaction, the reaction mixture was filtered anddried under reduced pressure and the purified by reverse phasepurification to afford the desired compound (34 mg, 44.5%) as whitesolid. LCMS: 504.58 (M+1⁺; ¹H NMR (400 MHz, DMSO-d₆): δ 10.31 (br s, 1H)9.23 (br s, 2H) 8.73-8.87 (m, 1H), 7.97-8.05 (m, 1H), 7.87 (d, J=8.3 Hz,1H), 7.65 (br s, 1H), 7.48 (d, J=8.3 Hz, 1H), 6.83 (d, J=8.7 Hz, 1H),4.36 (br s, 1H) 4.14-4.26 (m, 2H), 3.26 (br s, 2H), 1.52 (s, 6H), 1.33(d, J=6.5 Hz, 6H), 1.08 (br s, 4H).

Example S-24: Synthesis of2-ethyl-1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(Compound No. 1.261)

Step-1: Synthesis of2-ethyl-1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:To a stirred solution of2-ethyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(500 mg, 2.37 mmol, 1.0 eq) and2-(6-bromo-3-fluoropyridin-2-yl)propan-2-ol (556.6 mg, 2.37 mmol, 1.0eq) in dioxane (10 mL) was added potassium carbonate (657.6 mg, 4.75mmol, 2.0 eq) and the resulting mixture was purged with nitrogen for 10min followed by addition of copper iodide (90.5 mg, 0.47 mmol, 0.2 eq),and N,N′-dimethylethylenediamine (DMEDA) (0.09 mL, 0.951 mmol, 0.4 eq)and again purged with nitrogen for 10 min, stirred at 130° C. overnight.After completion of reaction, the reaction mixture was diluted withwater and extracted with EtOAc (50 mL×2). The combined organic layer waswashed with water (50 mL) and brine solution (50 mL), dried overanhydrous sodium sulfate and concentrated under reduced pressure toafford crude product, which was purified by flash chromatography [silicagel 100-200 mesh; elution 0-50% EtOAc in hexane] to afford the desiredproduct (520 mg, 60.2%) as brown liquid. LCMS: 364.44 (M+1)⁺.

Step-2: Synthesis of tert-butyl6-((2-ethyl-1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate):To a stirred solution of2-ethyl-1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one (250 mg, 0.68 mmol, 1.0 eq) in toluene (5.0 mL)was added m-CPBA (304 mg, 1.37 mmol, 2.0 eq) and allowed to stir at RTfor 60 minutes. Tert-butyl6-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (170.8 mg, 0.68 mmol,1.0 eq) and Na₂CO₃ (291.2 mg, 2.11 mmol, 4.0 eq) were added and allowedto stir at RT overnight. After completion of reaction, the reactionmixture was diluted with water and extracted with EtOAc (50 mL×2). Thecombined organic layer was washed with water (50 mL) and brine solution(50 mL), dried over anhydrous sodium sulfate and concentrated underreduced pressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-50% EtOAc in hexane]to afford the desired compound (97 mg, 25.0%) as light brown solid.LCMS: 564.7 (M+1)⁺.

Step-3: Synthesis of2-ethyl-1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-((1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:Tert-butyl6-((2-ethyl-1-(5-fluoro-6-(2-hydroxypropan-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate(90 mg, 0.16 mmol, 1.0 eq) was dissolved in dioxane (1 mL), followed bydropwise addition of 4.0 M dioxane in HCl (10 mL) and allowed to stir atRT for 2 h. After completion of reaction, the reaction mixture wasfiltered and dried under reduced pressure; purified product was obtainedby reverse phase purification (34 mg, 44.6%) as an off-white solid.LCMS: 464.58 (M+1)⁺; ¹H NMR (400 MHz, DMSO-d₆): δ10.19 (br s, 1H) 8.84(s, 1H) 7.87-8.06 (m, 2H) 7.51 (br s, 1H) 7.38 (d, J=8.3 Hz, 1H) 7.03(d, J=8.3 Hz, 1H) 5.25 (s, 1H) 4.01 (d, J=7.0 Hz, 2H) 3.86 (s, 2H) 2.98(t, J=5.7 Hz, 2H) 2.67 (d, J=5.3 Hz, 2H) 1.41-1.65 (s, 6H) 1.17-1.29 (m,2H) 0.97 (t, J=7.0 Hz, 3H).

Example S-25: Synthesis of1-(6-(6-((4,4-dimethyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropane-1-carbonitrile(Compound No. 1.40)

Step-1: Synthesis of tert-butyl7-((1-(6-(1-cyanocyclopropyl)pyridin-2-yl)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-4,4-dimethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of1-(6-(2-ethyl-6-(methylthio)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropane-1-carbonitrile(100 mg, 0.28 mmol, 1.0 eq) in toluene (3.0 mL) was added m-CPBA (134mg, 0.56 mmol, 2.0 eq) and allowed to stir at RT for 1 h. Tert-butyl7-amino-4,4-dimethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (78 mg,0.28 mmol, 1.0 eq) and Na₂CO₃ (120 mg, 1.132 mmol, 4.0 eq) were addedand allowed to stir at RT overnight. After completion of reaction, thereaction mixture was diluted with water and extracted with EtOAc (50mL×2). The combined organic layer was washed with water (50 mL) andbrine solution (50 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure to afford crude product, which waspurified by flash chromatography [silica gel 100-200 mesh; elution 0-50%EtOAc in hexane] to afford the desired compound (51 mg, 30.9%) as lightyellow solid. LCMS: 581.29 (M+1)⁺.

Step-2: Synthesis of1-(6-(6-((4,4-dimethyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropane-1-carbonitrile:Tert-butyl7-((1-(6-(1-cyanocyclopropyl)pyridin-2-yl)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-4,4-dimethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate(51 mg, 0.087 mmol, 1.0 eq) was dissolved in dioxane (1 mL), followed bydropwise addition of 4.0 M-HCl in dioxane (1 mL) and allowed to stir atRT for 2 h. After completion of reaction, the reaction mixture wasfiltered and dried under reduced pressure, crude was given for preppurification to afford the desired compound (22 mg, 45.2%) as whitesolid. LCMS: 481.24 (M+1)⁺; ¹H NMR (400 MHz, DMSO-d₆): δ 10.36 (br s,1H) 9.11 (br s, 2H) 8.88 (s, 1H) 8.14 (t, J=7.7 Hz, 1H) 7.93 (d, J=7.8Hz, 1H) 7.63 (br s, 1H) 7.49-7.60 (m, 1H) 7.45 (d, J=8.7 Hz, 1H) 4.26(br s, 2H) 3.98 (m, J=7.45 Hz, 2H) 3.24 (br s, 2H) 1.81-1.91 (m, 2H)1.63-1.75 (m, 2H) 1.35 (s, 6H) 0.97 (t, J=7.02 Hz, 3H).

Example S-26: Synthesis of1-(6-(3-oxo-6-(1,2,3,4-tetrahydroisoquinolin-7-ylamino)-2-(2,2,2-trifluoroethyl)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropanecarbonitrile(Compound No. 1.32)

Step-1: Synthesis of1-(6-(6-(methylthio)-3-oxo-2-(2,2,2-trifluoroethyl)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropanecarbonitrile:To a stirred solution of6-(methylthio)-2-(2,2,2-trifluoroethyl)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one(264 mg, 1.0 mmol, 1.0 eq) and1-(6-bromopyridin-2-yl)cyclopropane-1-carbonitrile (245 mg, 1.1 mmol,1.0 eq) in dioxane (10 mL) was added potassium carbonate (276 mg, 2.0mmol, 2.0 eq) and the resulting mixture was purged with nitrogen for 10min followed by addition of copper iodide (38 mg, 0.2 mmol, 0.2 eq), andN,N′-dimethylethylenediamine (DMEDA) (0.05 mL, 0.4 mmol, 0.4 eq) andagain purged with nitrogen for 10 min, then stirred at 130° C.overnight. After completion of reaction, the reaction mixture wasdiluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layer was washed with water (50 mL) and brine solution (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-50% EtOAc in hexane]to afford the desired product, (220 mg, 54.2%) as light brown solid.LCMS: 407.3 (M+1)⁺.

Step-2: Synthesis of tert-butyl7-(1-(6-(1-cyanocyclopropyl)pyridin-2-yl)-3-oxo-2-(2,2,2-trifluoroethyl)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-ylamino)-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of1-(6-(6-(methylthio)-3-oxo-2-(2,2,2-trifluoroethyl)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropanecarbonitrile (102 mg,0.25 mmol, 1.0 eq) in toluene (2.0 mL) was added m-CPBA (122 mg, 0.5mmol, 2.0 eq) and allowed to stir at RT for 60 minutes. Tert-butyl7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (75 mg, 0.31 mmol, 1.2eq) and Na₂CO₃ (106 mg, 1.0 mmol, 4.0 eq) were added and allowed to stirat RT overnight. After completion of reaction, the reaction mixture wasdiluted with water and extracted with EtOAc (50 mL×2). The combinedorganic layer was washed with water (50 mL) and brine solution (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure to afford crude product, which was purified by flashchromatography [silica gel 100-200 mesh; elution 0-50% EtOAc in hexane]to afford the desired compound, (100 mg, 72%) as off-white solid. LCMS:607.4 (M+1)⁺.

Step-3: Synthesis of1-(6-(3-oxo-6-(1,2,3,4-tetrahydroisoquinolin-7-ylamino)-2-(2,2,2-trifluoroethyl)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropanecarbonitrile:Tert-butyl7-(1-(6-(1-cyanocyclopropyl)pyridin-2-yl)-3-oxo-2-(2,2,2-trifluoroethyl)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-ylamino)-3,4-dihydroisoquinoline-2(1H)-carboxylate(100 mg, 0.18 mmol, 1.0 eq) was dissolved in dioxane (1 mL), followed bydropwise addition of 4.0 M-HCl in dioxane (1 mL) and allowed to stir atRT for 4 h. After completion of reaction, the reaction mixture wasfiltered, dried under reduced pressure and triturated with diethyl etherto give white solid. To this solid was added aq. NaHCO₃ (10 mL) andproduct was extracted in to DCM. Organic layer was dried over anhydroussodium sulfate and concentrated under reduced pressure to afford thedesired product as an off-white solid (20 mg, 22%). LCMS: 507.2 (M+1)⁺;¹H NMR (400 MHz, DMSO-d₆): δ10.43 (br s, 1H) 8.95 (s, 1H) 8.11 (t, J=7.8Hz, 1H) 7.91 (br s, 1H) 7.53 (d, J=7.8 Hz, 2H) 7.33 (br s, 1H) 7.04 (d,J=8.3 Hz, 1H) 4.91 (d, J=8.7 Hz, 2H) 3.85 (br s, 2H) 2.96 (d, J=6.1 Hz,2H) 2.67 (d, J=1.75 Hz, 2H) 1.79-1.89 (m, 2H) 1.63-1.76 (m, 2H).

Example S-27: Synthesis of1-(6-(6-(1,1-dimethyl-1,2,3,4-tetrahydroisoquinolin-6-ylamino)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropanecarbonitrile(Compound No. 1.39)

Step-1: Synthesis of tert-butyl6-(1-(6-(1-cyanocyclopropyl)pyridin-2-yl)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-ylamino)-1,1-dimethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of1-(6-(2-ethyl-6-(methylthio)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropanecarbonitrile (125 mg,0.35 mmol, 1.0 eq) in toluene (2.0 mL) was added m-CPBA (172 mg, 0.7mmol, 2.0 eq) and allowed to stir at RT for 60 minutes. Tert-butyl6-amino-1,1-dimethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (107 mg,0.39 mmol, 1.1 eq) and Na₂CO₃ (149 mg, 1.4 mmol, 4.0 eq) were added andallowed to stir at RT overnight. After completion of reaction, thereaction mixture was diluted with water and extracted with EtOAc (50mL×2). The combined organic layer was washed with water (50 mL) andbrine solution (50 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure to afford crude product, which waspurified by flash chromatography [silica gel 100-200 mesh; elution 0-50%EtOAc in hexane] to afford the desired compound, (110 mg, 54.2%) aslight yellow solid. LCMS: 581.2 (M+1)⁺.

Step-2: Synthesis of1-(6-(6-(1,1-dimethyl-1,2,3,4-tetrahydroisoquinolin-6-ylamino)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropanecarbonitrile:Tert-butyl6-(1-(6-(1-cyanocyclopropyl)pyridin-2-yl)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-ylamino)-1,1-dimethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate(110 mg, 0.19 mmol, 1.0 eq) was dissolved in dioxane (2 mL), followed bydropwise addition of 4.0 M-HCl in dioxane (2 mL) and allowed to stir atRT for 2 h. After completion of reaction, solvent was evaporated,triturated with diethyl ether (3×5 mL) and dried to give the desiredcompound (76 mg, 83.3%) as an off-white solid. LCMS: 481.5 (M+1)⁺; ¹HNMR (400 MHz, DMSO-d₆): δ10.34 (br s, 1H) 9.62 (br s, 2H) 8.77-8.94 (s,1H) 8.17 (t, J=7.8 Hz, 1H) 7.91 (d, J=7.8 Hz, 1H) 7.68 (br s, 1H)7.45-7.56 (m, 2H) 7.36 (d, J=8.7 Hz, 1H) 3.98 (q, J=6.8 Hz, 2H) 3.40 (brs, 2H) 3.04 (t, J=6.1 Hz, 2H) 1.81-1.93 (m, 2H) 1.58-1.77 (m, 8H) 0.97(t, J=7.0 Hz, 3H).

Example S-28: Synthesis of1-(6-(6-(2′,3′-dihydro-1H-spiro[cyclopropane-1,4′-isoquinoline]-7′-ylamino)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropanecarbonitrile(Compound No. 1.42)

Step-1: Synthesis of tert-butyl7′-(1-(6-(1-cyanocyclopropyl)pyridin-2-yl)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-ylamino)-1′H-spiro[cyclopropane-1,4′-isoquinoline]-2′(3′H)-carboxylate:To a stirred solution of1-(6-(2-ethyl-6-(methylthio)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropanecarbonitrile (125 mg,0.35 mmol, 1.0 eq) in toluene (2.0 mL) was added m-CPBA (172 mg, 0.7mmol, 2.0 eq) and allowed to stir at RT for 60 minutes. Tert-butyl7′-amino-1′H-spiro[cyclopropane-1,4′-isoquinoline]-2′(3′H)-carboxylate(106 mg, 0.39 mmol, 1.1 eq) and Na₂CO₃ (149 mg, 1.4 mmol, 4.0 eq) wereadded and allowed to stir at RT overnight. After completion of reaction,the reaction mixture was diluted with water and extracted with EtOAc (50mL×2). The combined organic layer was washed with water (50 mL) andbrine solution (50 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure to afford crude product, which waspurified by flash chromatography [silica gel 100-200 mesh; elution 0-50%EtOAc in hexane] to afford the desired compound, (90 mg, 44.6%) as lightyellow solid. LCMS: 579.2 (M+1)⁺.

Step-2: Synthesis of1-(6-(6-(2′,3′-dihydro-1′H-spiro[cyclopropane-1,4′-isoquinoline]-7′-ylamino)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropanecarbonitrile:Tert-butyl7′-(1-(6-(1-cyanocyclopropyl)pyridin-2-yl)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-ylamino)-1′H-spiro[cyclopropane-1,4′-isoquinoline]-2′(3′H)-carboxylate(90 mg, 0.16 mmol, 1.0 eq) was dissolved in dioxane (1 mL), followed bydropwise addition of 4.0 M-HCl in dioxane (1 mL) and allowed to stir atRT for 4 h. After completion of reaction, triturated with diethyl ether(3×5 mL) and dried to give the desired compound (31 mg, 40.5%) as anoff-white solid. LCMS: 479.5 (M+1)⁺; ¹H NMR (400 MHz, DMSO-d₆): δ10.34(br s, 1H) 9.36 (br s, 2H) 8.88 (s, 1H) 8.16 (t, J=7.9 Hz, 1H) 7.92 (d,J=7.9 Hz, 1H) 7.67 (br s, 1H) 7.45-7.56 (m, 2H) 6.84 (d, J=8.7 Hz, 1H)4.37 (br s, 2H) 3.89-4.03 (m, 2H) 3.26 (br s, 2H) 1.77-1.92 (m, 2H)1.60-1.76 (m, 2H) 1.09 (d, J=4.4 Hz, 4H) 0.97 (t, J=6.8 Hz, 3H).

Example S-29: Synthesis of1-(4-(6-((1,1-dimethyl-1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropane-1-carbonitrile(Compound No. 1.63)

Step-1: Synthesis of tert-butyl6-((1-(2-(1-cyanocyclopropyl)pyridin-4-yl)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-1,1-dimethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of1-(4-(2-ethyl-6-(methylthio)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropane-1-carbonitrile(100 mg, 0.28 mmol, 1.0 eq) in toluene (3 mL) was added m-CPBA (126 mg,0.56 mmol, 2.0 eq) and allowed to stir at RT for 60 minutes. tert-butyl6-amino-1,1-dimethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (78.4 mg,0.28 mol, 1.0 eq) and Na₂CO₃ (120 mg, 1.13 mmol, 4.0 eq) were added andallowed to stir at RT for overnight. After completion of reaction, thereaction mixture was diluted with water and extracted with EtOAc (50mL×2). The combined organic layer was washed with water (50 mL), brinesolution (50 mL), dried over anhydrous sodium sulfate and concentratedunder reduced pressure to afford crude product, which was purified byflash chromatography [silica gel 100-200 mesh; elution 0-50% EtOAc inhexane] to afford the titled compound (70 mg, 42.5%). LCMS: 581.69(M+1)⁺.

Step-2: Synthesis of1-(4-(6-((1,1-dimethyl-1,2,3,4-tetrahydroisoquinolin-6-yl)amino)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-2-yl)cyclopropane-1-carbonitrile:tert-butyl6-((1-(2-(1-cyanocyclopropyl)pyridin-4-yl)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-1,1-dimethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate(70 mg, 0.12 mmol, 1.0 eq) was dissolved in dichloromethane (1 mL),followed by dropwise addition of TFA (0.5 mL) and allowed to stir at rtfor 1 h. After completion of reaction, the reaction mixture was filteredand dried under reduced pressure and triturated with diethyl ether toafford the titled compound (4 mg, 6.3%). LCMS: 481.58 (M+1)⁺; ¹H NMR(400 MHz, DMSO-d₆): δ10.43 (br s, 1H) 8.84-8.95 (m, 1H) 8.69 (d, J=5.2Hz, 1H) 7.48-7.67 (m, 4H) 7.37 (d, J=8.7 Hz, 1H) 3.70-3.86 (m, 2H) 3.39(br s, 2H), 2.97 (br s, 2H) 1.70-1.95 (m, 4H), 1.61 (s, 6H), 0.98 (t,J=7.2 Hz, 3H).

Example S-30: Synthesis of2-(6-(6-(1,1-dimethyl-1,2,3,4-tetrahydroisoquinolin-6-ylamino)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-5-fluoropyridin-2-yl)-2-methylpropanenitrile(Compound No. 1.335)

Step-1: Synthesis of2-(6-(2-ethyl-6-(methylthio)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-5-fluoropyridin-2-yl)-2-methylpropanenitrile:To a stirred solution of2-(6-bromo-5-fluoropyridin-2-yl)-2-methylpropanenitrile (300 mg, 1.23mmol, 1.0 eq) and2-ethyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(259 mg, 1.23 mmol, 1.0 eq) in (10 mL) of dioxane were added Potassiumcarbonate (341.2 mg, 2.46 mmol, 2.0 eq) and the resulting mixture waspurged with nitrogen for 10 min followed by addition of copper iodide(47 mg, 0.24 mmol, 0.2 eq), and N,N′-dimethylethylenediamine (DMEDA)(0.05 mL, 0.49 mmol, 0.4 eq) and again purged with nitrogen for 10 min,stirred at 900 C for overnight. After completion of reaction, thereaction mixture was diluted with water and extracted with EtOAc (50mL×2). The combined organic layers were washed with water (50 mL) brinesolution (50 mL), dried over anhydrous sodium sulphate and concentratedunder reduced pressure to afford crude product, which was purified byflash chromatography to afford the titled compound (106 mg, 23.1%).LCMS: 372.4 (M+1)⁺.

Step-2: Synthesis of tert-butyl6-((1-(6-(2-cyanopropan-2-yl)-3-fluoropyridin-2-yl)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-1,1-dimethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate:To a stirred solution of2-(6-(2-ethyl-6-(methylthio)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-5-fluoropyridin-2-yl)-2-methylpropanenitrile(106 mg, 0.28 mmol, 1.0 eq) in (3.0 mL) of toluene was added m-CPBA(126.4 mg, 0.56 mmol, 2.0 eq) and allowed to stir at RT for 1 h.tert-butyl6-amino-1,1-dimethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (79 mg,0.28 mmol, 1.05 eq) and Na₂CO₃ (120 mg, 1.13 mmol, 4.0 eq) were addedand allowed to stir at RT for overnight. After completion of reaction,the reaction mixture was diluted with water (50 mL) and extracted withEtOAc (50 mL×2). The combined organic layer was washed with water (50mL), brine solution (50 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure to afford crude product, which waspurified by flash chromatography [silica gel 100-200 mesh; elution 0-50%EtOAc in hexane] afford the titled compound (40 mg, 23.4%). LCMS: 600.7(M+1)⁺.

Step-3: Synthesis of2-(6-(6-(1,1-dimethyl-1,2,3,4-tetrahydroisoquinolin-6-ylamino)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-5-fluoropyridin-2-yl)-2-methylpropanenitrile:tert-butyl6-((1-(6-(2-cyanopropan-2-yl)-3-fluoropyridin-2-yl)-2-ethyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-1,1-dimethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate(40 mg, 0.06 mmol, 1.0 eq) was dissolved in dioxane (1 mL), followed bydropwise addition of 4.0 M HCl in dioxane (1 mL) and allowed to stir atRT for 1 h. After completion of reaction, the reaction mixture wasfiltered and dried under reduced pressure to afford the titled compound(22 mg, 60.5%). LCMS: 501.5 (M+1)⁺; ¹H NMR (400 MHz, DMSO-d₆): δ10.24(br s, 1H) 8.87 (s, 1H) 8.26 (br s, 1H) 8.13-8.24 (m, 2H) 8.09 (d, J=6.1Hz, 1H) 7.51 (br s, 1H) 7.45 (d, J=8.3 Hz, 1H) 7.27 (d, J=8.7 Hz, 1H)3.95-4.09 (m, 2H) 3.10 (br s, 2H) 2.77 (br s, 2H) 1.76 (s, 6H) 1.43 (s,6H) 0.98 (t, J=7.0 Hz, 3H).

The compounds disclosed therein are prepared according to theexperimental details exemplified in Examples S1-S30 and Scheme 1 toScheme 5, using the appropriate starting materials and reagents.

BIOLOGICAL EXAMPLES Example B1. WEE1 IC₅₀ Determination

IC₅₀ values of compounds against WEE1 kinase enzyme were determined byLanthaScreen™ Terbium Labeled TR-FRET assay. Kinase assays wereperformed in 1X kinase buffer (#PV6135, Invitrogen, Life TechnologiesGrand Island, N.Y.) where total reaction volume was 10 p L in low-volume384-well plates (#4511, Corning). Serially diluted compounds (3-fold)were incubated with WEE1 Enzyme (1 nM) (#PR7373A, Invitrogen, LifeTechnologies, Grand Island, N.Y.) for 10 min; a mixture of ATP (10 μM)(#A1852, Sigma, St. Louis, Mo.) and fluorescent-PolyGT substrate (200nM) (#PV3610, Invitrogen, Life Technologies Grand Island, N.Y.) wasadded and incubated in dark at room temperature for 1 h. After 1 h, 10μL stop solution containing Terbium labeled antibody (4 nM) (#PV3529,Invitrogen, Life Technologies Grand Island, N.Y.) and EDTA (#E5134,Sigma, St. Louis, Mo.) (20 mM) in TR-FRET dilution buffer (# PV3574,Invitrogen, Life Technologies Grand Island, N.Y.) was added. Readingswere taken in a Synergy Neo Plate reader (BioTek, Winooski, Vt.) atsingle excitation of 340 nm and dual emission at 495 nm and 520 nmrespectively.

The % activity of test samples was calculated as(Sample−Min)*100/(Max−Min). [Max: DMSO control, complete reaction withenzyme & DMSO and Min: No enzyme & DMSO]. Percent inhibition (100−%activity) was fitted to the “four-parameter logistic model” in XLfit fordetermination of IC₅₀ values. The results are shown in Table 2.

TABLE 2 Compound No. Wee1 IC₅₀ (μM) 1.1 0.006 1.2 0.036 1.4 0.013 1.50.005 1.32 0.008 1.37 0.013 1.38 0.004 1.39 0.001 1.40 0.004 1.42 0.0011.43 0.009 1.44 0.003 1.45 0.010 1.63 0.018 1.67 0.015 1.86 0.002 1.910.003 1.188 0.027 1.249 0.016 1.250 0.008 1.251 0.007 1.254 0.140 1.2610.009 1.262 0.008 1.321 0.013 1.322 0.007 1.335 0.005 1.349 0.384 1.4640.049 1.465 0.045

Example B2. PKMYT1 IC₅₀ Determination

Inhibition of PKMYT1 kinase activity by test compounds was measured bythe HotSpot Kinase Assay at Reaction Biology Corporation (Malvern, Pa.).Briefly, Myelin Basic Protein substrate was prepared in Reaction Buffer(20 mM Hepes (pH 7.5), 10 mM MgCl₂, 1 mM EGTA, 0.01% Brij35, 0.02 mg/mLBSA, 0.1 mM Na₃VO₄, 2 mM DTT, 1% DMSO). PKMYT1 kinase was delivered intothe substrate solution and gently mixed. Test compounds in 100% DMSOwere added into the kinase reaction mixture by Acoustic technology(Echo550; nanoliter range) and incubated for 20 min at room temperature.³³P-ATP was delivered into the reaction mixture to initiate thereaction. Reactions were carried out at 10 μM ATP. After a 2 hourincubation at room temperature, kinase activity was detected by P81filter-binding method. Compounds were tested in 10-dose IC₅₀ mode with a3-fold serial dilution. A nonlinear regression model with a sigmoidaldose response and variable slope within GraphPad Prism (GraphPadSoftware, San Diego, Calif.) was used to calculate the IC₅₀ value ofindividual test compounds. The results are shown in Table 3.

TABLE 3 Compound No. PKMYT1 IC₅₀ (μM) 1.1 6.57 1.2 10.2 1.4 17.8 1.5 5.51.32 0.767 1.37 0.691 1.38 0.223 1.40 0.121 1.43 5.63 1.44 1.17 1.451.54 1.67 2.9 1.86 4.2 1.91 6.92 1.188 2.24 1.249 20.1 1.250 13.5 1.2516.69 1.254 3.56 1.261 4.54 1.262 16.8 1.321 11.2 1.322 7.49 1.349 19.91.464 28.8 1.465 >30

Example B3. Determination of Potency of Compounds in Cytotoxicity Assayin A427 Cell Line

A427 (HTB-53; ATCC), a lung epithelial cell line, was seeded in medium(MEM, 41090101; Gibco) at a cell count of 1500 cells per 100 μL per wellin a 96 well edge plate (167425; ThermoFisher). Cells were allowed togrow at 37° C. for 24 hr in 5% CO₂ environment (culture conditions) in aNuaire incubator (humidified). Serially diluted test compounds (100 μL)within the desired testing concentration ranges were added to theculture plate and the cells were further incubated in culture conditionsfor 72 hr. The experiment was terminated at the designated incubationtime by replacing the medium with 100 μL of 1 mM of resazurin (R7017;Sigma) prepared in culture medium, and the plates were further incubatedin culture conditions for 4-6 hr. Fluorescence was recorded using amultimodal plate reader (Biotek Synergy Neo) at an excitation wavelengthof 535 nm and emission wavelength of 590 nm to obtain relativefluorescence units. Data were analysed as follows: the backgroundfluorescence (blank containing only medium) value was subtracted fromeach reading and normalized with the vehicle control (DMSO treatedcells) to obtain percent survival/proliferation. Percent survival wassubtracted from 100 to get the percent inhibition of proliferation whichwas used to calculate IC₅₀ values. Potency of compounds in other celllines (such as A549, AsPc-1, Panc 10.05, A172, U-87MG) may be determinedin an analogous manner. The results are shown in Table 4.

TABLE 4 Compound No. A427 IC₅₀ (μM) 1.1 3.740 1.2 3.410 1.4 1.410 1.57.599 1.32 0.870 1.37 0.660 1.38 0.401 1.39 0.510 1.40 0.440 1.42 0.2551.43 1.478 1.44 0.892 1.45 2.179 1.63 17.160 1.67 2.575 1.86 0.685 1.910.565 1.188 2.171 1.249 0.592 1.250 2.690 1.251 12.355 1.254 0.765 1.2610.900 1.262 1.455 1.321 3.537 1.322 2.281 1.335 4.733 1.349 0.490 1.46424.621 1.465 >30

Example B4. Determination of Potency of Compounds in Cell ProliferationAssay in Selected Cancer Cell Lines and Cellular PD Effects

The effects of test compounds are studied in additional cell lines withvarious histotypes, such as LoVo colorectal adenocarcinoma, NCI-H460large-cell lung carcinoma, HCT-116 colorectal carcinoma, and A2780ovarian cancer cells. The cancer cells are harvested during thelogarithmic growth period and counted. Cell concentrations are adjustedto the appropriate number with suitable medium, and 90 μL cellsuspensions are added to 96-well plates. After cells are seeded, theplates are shaken gently to distribute cells evenly and incubated at 37°C., 5% CO₂ on day 1.

Cells are treated with test compounds at 9 concentrations within adesired concentration range (e.g. 1.5 nM-10 μM) on day 2 by seriesdiluting the test compound stock solution (10 mM in DMSO) with culturemedium. Cell viability is assessed by Cell Titer-Glo® as recommended byPromega (Cat. No.: G7572, Promega) typically 72 h post-treatment.

Cell viability data are plotted using GraphPad Prism (version 5,GraphPad Software, Inc., San Diego, Calif.). In addition, a nonlinearregression model with a sigmoidal dose response and variable slopewithin GraphPad Prism is used to calculate the IC₅₀ value of individualtest compounds.

Test compounds may be studied in the same and/or other cancer cell lineswith varying sensitivities to reported Wee1 inhibiting compounds usingsimilar proliferation methods with possible variations in cell seedingdensities and/or incubation durations.

Example B5. Determination of Potency of Compounds by Assay of CellularPD Effects

Phospho-CDC2 and γ-H2AX are two clinically relevant biomarkersassociated with Wee1 inhibition. CDC2Y15 phosphorylation in cells wasreported to be abolished by Wee1 inhibitors (Gavory G et. al., AlmacDiscovery, AACR poster, 2016). γ-H2AX, a DNA double-strand break marker,was upregulated by Wee1 treatment in Wee1 sensitive cell lines (GuertinA D et al., Molecular Cancer Therapeutics, 2013). The effects ofselected test compounds on pCDC2 and γ-H2AX are assessed in selectedcancer cell lines post 24 or 48 hr treatment using Western blottingmethods with selective antibodies (Guertin A D et al., Molecular CancerTherapeutics, 2013).

Changes in the levels of phospho-CDC2 following treatment of cells withtest compounds were assessed by enzyme-linked immunosorbent assay(ELISA). A427 cells or AsPC-1 cells were plated in 6-well plates andcultured for 24 hr to approximately 80-90% confluency. Medium wasreplaced, and the cells were treated with the vehicle control or thetest compound at several different concentrations. After incubation oftreated cells in cell culture conditions for a specified time (e.g., 24hr), cells were rinsed with ice-cold PBS and lysed in 1X cell lysisbuffer containing protease inhibitors and phosphatase inhibitors. Thecells were scraped from the plate with a cell scraper after a briefincubation on ice and transferred to a centrifuge tube, and thensubjected to three freeze-thaw cycles in liquid nitrogen and a 37° C.water bath for further lysis. The lysates were centrifuged to pelletcell debris (using, for example, a 10 min centrifugation of 2000×g at 4°C.) and the supernatants transferred to fresh tubes on ice. The proteinconcentrations of the samples were estimated by the Bradford method orequivalent. The ELISA was carried out with the PathScan® Phospho-CDC2(Tyr15) Sandwich ELISA Kit (Cat. #7176, Cell Signaling Technology,Danvers, Mass.) according to the manufacturer's instructions. Resultsare shown in Table 5.

TABLE 5 A427 phospho- AsPC-1 phospho- Compound No. CDC2 IC₅₀ (μM) CDC2IC₅₀ (μM) 1.1 0.339 ND 1.2 0.952 ND 1.4 0.414 ND 1.38 0.176 ND 1.430.320 ND 1.45 0.066 0.285 1.249 0.230 ND ND: Not Determined

Changes in the levels of phospho-CDC2 are alternatively or additionallyanalyzed by Western blotting of the samples using a primary antibody tophospho-CDC2 such as phospho-CDC2 (Tyr15) (10A11) rabbit mAb (Cat.#4539, Cell Signaling Technology) or rabbit polyclonal anti-CDK1(phospho Y15) antibody (Cat. #ab47594, Abcam, Cambridge, UnitedKingdom).

Example B6. Evaluation of Test Compound in Mouse Xenograft Models

To examine the in vivo antitumor activity of test compound (as a singleagent and in combination with other agents such as gemcitabine,nab-paclitaxel and temozomide), tumor growth experiments are performedin a cell line xenograft model and/or a PDX model. The cell line ischosen based on the in vitro studies described above. The PDX model tobe used is established from a tumor taken directly from a patient with,for example, pancreatic ductal adenocarcinoma (PDAC) or glioblastoma.

Cells or tumor chucks are implanted subcutaneously into the flanks ofnude mice and allowed to grow until the tumor size reaches 200 mm³.Tumors are measured using a caliper and tumor volumes calculated usingthe formula: Tumor volume=(a×b²/2) where ‘b’ is the smallest diameterand ‘a’ is the largest diameter. Once the established tumors reachapproximately 200 mm³, the mice are then stratified into treatmentgroups. The treatment groups are, for example: vehicle control,gemcitabine+nab-paclitaxel, test compound alone,gemcitabine+nab-paclitaxel+test compound at 10 mice per group. Thetreatment groups are alternatively, for example: vehicle control,temozolomide, test compound alone, temozolomide+test compound. The exacttreatment groups, drug dose, and dosing schedule are determinedspecifically for each study according to standard practice. Tumor growthis monitored, and volume recorded at regular intervals. When theindividual tumor of each mouse reaches an approximate end-point (tumorvolume>1,500 mm³), the mouse is sacrificed with regulated CO₂. The tumorgrowth inhibition (TGI) is calculated by comparing the control group'stumor measurements with the other study groups once the predeterminedendpoint is reached in the control group. Alternatively, cells areimplanted orthotopically and overall survival is measured.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is apparent to those skilled in the art that certainminor changes and modifications will be practiced in light of the aboveteaching. Therefore, the description and examples should not beconstrued as limiting the scope of the invention.

1. A compound of Formula (I):

or a stereoisomer or tautomer thereof, or a pharmaceutically acceptablesalt of any of the foregoing, wherein: Y is hydrogen or R⁴; m is 0, 1,2, or 3; n is 0, 1, 2, 3, or 4; R¹ is independently F, Cl, or methyl; R²is C₁-C₆ alkyl, C₃-C₆ cycloalkyl or —(C₁-C₃ alkylene)CF₃; R³ is

 wherein:

 indicates an aromatic ring; M¹ is CH or CR³; M² is CH, CR^(3b), N, orabsent; M³ is CH, CR^(3b), N, O, or S; M⁴ is CH, CR³b, N, O, or S,provided that: (1) when M⁴ is O or S and M² is absent, then M³ is CH,CR^(3b) or N, and (2) when M³ is O or S and M² is absent, then M⁴ is CH,CR^(3b) or N; R^(3a) is C₃-C₆ cycloalkyl optionally substituted by C₁-C₆haloalkyl or —CN, or C₁-C₆ alkyl optionally substituted by halogen, —OHor —CN, provided that when R^(3a) is C₁-C₆ alkyl optionally substitutedby halogen, —OH or —CN, then at least one of M¹, M², M³, and M⁴ isCR^(3b); R^(3b) is halogen or —CN; each R⁴ is independently oxo, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, halogen, —C(O)R¹⁷, —C(O)OR¹⁷,—C(O)NR¹⁷R¹⁸, —CN, —Si(C₁-C₆ alkyl)₃, —OR¹⁷, —NR¹⁷R¹⁸, —OC(O)NR¹⁷R¹⁸,—NR¹⁷C(O)R¹⁸, —S(O)₂R¹⁷, —NR¹⁷S(O)₂R¹⁸, —S(O)₂NR¹⁷R¹⁸, C₃-C₆ cycloalkyl,3- to 6-membered heterocyclyl, —(C₁-C₃ alkylene)CN, —(C₁-C₃alkylene)OR¹⁷, —(C₁-C₃ alkylene)NR¹⁷R¹⁸, —(C₁-C₃ alkylene)CF₃, —(C₁-C₃alkylene)C(O)R¹⁷, —(C₁-C₃ alkylene)C(O)NR¹⁷R¹⁸, —(C₁-C₃alkylene)NR¹⁷C(O)R¹⁸, —(C₁-C₃ alkylene)S(O)₂R¹⁷, —(C₁-C₃alkylene)NR¹⁷S(O)₂R¹⁸, —(C₁-C₃alkylene)S(O)₂NR¹⁷R¹⁸, —(C₁-C₃alkylene)(C₃-C₆ cycloalkyl) or —(C₁-C₃ alkylene)(3- to 6-memberedheterocyclyl), wherein each R⁴ is independently optionally substitutedby halogen, oxo, —OR¹⁹, —NR¹⁹R²⁰, or —C(O)R¹⁹, or two R⁴, when bound tothe same carbon, are taken together with the carbon to which they areattached to form a C₃-C₆ cycloalkyl or 3- to 6-membered heterocyclyl,each is optionally substituted by R¹⁹; and each R¹⁷, R¹⁸, R¹⁹, and R²⁰is independently hydrogen, C₃-C₆ cycloalkyl, 3-6 membered heterocyclylor C₁-C₆ alkyl, each of which is optionally substituted by halogen, oxoor —OH, or R¹⁷ and R¹⁸ are taken together with the atom to which theyattached to form a 3-6 membered heterocyclyl optionally substituted byhalogen, oxo or —OH.
 2. The compound of claim 1, or a stereoisomer ortautomer thereof, or a pharmaceutically acceptable salt of any of theforegoing, wherein the compound is of Formula (II):


3. The compound of claim 1, or a stereoisomer or tautomer thereof, or apharmaceutically acceptable salt of any of the foregoing, wherein thecompound is of Formula (III):


4. The compound of claim 1, or a stereoisomer or tautomer thereof, or apharmaceutically acceptable salt of any of the foregoing, wherein R² isC₁-C₆alkyl.
 5. The compound of claim 4, or a stereoisomer or tautomerthereof, or a pharmaceutically acceptable salt of any of the foregoing,wherein R² is isopropyl or ethyl.
 6. The compound of claim 1, or astereoisomer or tautomer thereof, or a pharmaceutically acceptable saltof any of the foregoing, wherein R² is C₃-C₆cycloalkyl.
 7. The compoundof claim 6, or a stereoisomer or tautomer thereof, or a pharmaceuticallyacceptable salt of any of the foregoing, wherein R² is cyclopropyl. 8.The compound of claim 1, or a stereoisomer or tautomer thereof, or apharmaceutically acceptable salt of any of the foregoing, wherein R² is—(C₁-C₃ alkylene)CF₃.
 9. The compound of claim 8, or a stereoisomer ortautomer thereof, or a pharmaceutically acceptable salt of any of theforegoing, wherein R² is —CH₂CF₃.
 10. The compound of claim 1, or astereoisomer or tautomer thereof, or a pharmaceutically acceptable saltof any of the foregoing, wherein R³ is selected from the groupconsisting of:


11. The compound of claim 1, or a stereoisomer or tautomer thereof, or apharmaceutically acceptable salt of any of the foregoing wherein R^(3a)is C₃-C₆ cycloalkyl optionally substituted by C₁-C₆ haloalkyl or —CN.12. The compound of claim 1, or a stereoisomer or tautomer thereof, or apharmaceutically acceptable salt of any of the foregoing, wherein R^(3a)is C₁-C₆ alkyl optionally substituted by halogen, —OH or —CN.
 13. Thecompound of claim 1, or stereoisomer or tautomer thereof, or apharmaceutically acceptable salt of any of the foregoing, wherein R^(3a)is selected from the group consisting of:


14. The compound of claim 1, or a stereoisomer or tautomer thereof, or apharmaceutically acceptable salt of any of the foregoing, wherein R^(3b)is halogen.
 15. The compound of claim 1, or a stereoisomer or tautomerthereof, or a pharmaceutically acceptable salt of any of the foregoing,wherein R^(3b) is —CN.
 16. The compound of claim 1, or a stereoisomer ortautomer thereof, or a pharmaceutically acceptable salt of any of theforegoing, wherein R³ is selected from the group consisting of:

17-23. (canceled)
 24. The compound of claim 1, or a stereoisomer ortautomer thereof, or a pharmaceutically acceptable salt of any of theforegoing, wherein ring A, ring B, R¹, and R⁴ are taken together to forma moiety selected from the group consisting of:


25. A compound or a stereoisomer or tautomer thereof, or apharmaceutically acceptable salt of any of the foregoing, wherein thecompound is selected from the group consisting of


26. (canceled)
 27. A pharmaceutical composition comprising a compound ofclaim 1, or a stereoisomer or tautomer thereof, or a pharmaceuticallyacceptable salt of any of the foregoing, and a pharmaceuticallyacceptable carrier.
 28. A method of treating a cancer in an individualin need thereof comprising administering to the individual atherapeutically effective amount of a compound of claim 1, or astereoisomer or tautomer thereof, or a pharmaceutically acceptable saltof any of the foregoing. 29-41. (canceled)